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Volume 3 Number 2 July-December 201 3

Journal of Biology and Earth Sciences MNiSW 3 Index Copernicus 6.21 UIF 0.5238

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Contents i-ii iii-iv v-vi

Editorial sites Instructions for authors Contents

BIOLOGY B1 64-B1 68 Influence of synthetic and plant extracts on biochemical changes in the gonadal tissue of snail Lymnaea acuminata Vatsala Singh, J. N. Tiwari

B1 69-B1 75 Reptilia, Squamata, Colubridae, Elaphe radiata (Boie, 1 827): New range and habitat notes for Rajaji National Park, northwest India Ritesh Joshi

B1 76-B1 81 Impact of salicylic acid on biofilm formation by plant pathogenic bacteria Leonid Lagonenko, Alexander Lagonenko, Anatoly Evtushenkov

B1 82-B1 93 Indole-3-acetic acid (IAA) production by Streptomyces atrovirens isolated from rhizospheric soil in Egypt Mohamed Hemida Abd-Alla, El-Sayed A. El-Sayed, Abdel-Hamied M. Rasmey

B1 94-B205 Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae a review Sunil Kumar Singh, Shailendra K. Singh, Ajay Singh

B206-B21 3 The effects of soil heavy metals pollution and seasonal variations on gametogenesis and energy reserves of the land snail Eobania vermiculata Abdelmonem Mohamed Khalil

B21 4-B227 Vegetation analysis and species diversity in the desert ecosystem of coastal wadis of South Sinai, Egypt Fawzy Mahmoud Salama, Monier Mohamed Abd El-Ghani, Salah Mohamed El-Naggar, Mohamed Meftah Aljarroushi

B228-B235 In-vivo study of effects of dithiocarbamates fungicide (Mancozeb) and its metabolite ethylenethiourea (ETU) on fresh water fish Clarius batrachus Ajay Singh, Pallavi Srivastava

B236-B248 Seasonal variation of heavy metals accumulation in muscles of the African Catfish Clarias gariepinus and in River Nile water and sediments at Assiut Governorate, Egypt Hossam El-Din Mohamed Omar

B249-B254 Cisplatin and/or etoposide induces oxidative stress in testicular, hepatic and kidney tissues in male albino mice U. Kanchana Ganga, Battini Kishori, P. Sreenivasula Reddy

B255-B260 Trizole: a new fungicidal group induced chromosomal aberrations in Asian Catfish (Clarius batrachus) Pallavi Srivastava, Ajay Singh

B261 -B268 Ecotoxicological bioassays of the earthworms Allolobophora caliginosa Savigny and Pheretima hawayana Rosa treated with arsenate under laboratory conditions Abdelmonem Mohamed Khalil

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vi B269-B274 Insecticidal effect of methoprene on the pre-adult stages of almond moth, Ephestia cautella Walker (Lepidoptera: Pyralidae) Awanish Chandra, Shri Krishna Tiwari

B275-B285 Gibberellic acid ameliorates the adverse effects of acid mist and improved antioxidant defense, water status and growth of acid misted sunflower plants Suzan Sayed, Mohamed A. A. Gadallah

MEDICINE M48-M51

Molecular basis of human obesity - literature review

Agata Tomaszewska, Przemysław Kopczyński, Rafał Flieger, Anna Thielemann

EARTH SCIENCES E1 2-E1 7

Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India

E1 8-E38

Plutonites of Wadi Um Arka, Allaqi region, South Eastern Desert, Egypt: remote sensing and geochemical aspects

Poonam Bhadja, Ashokkumar Vaghela

Ezzat Abdel Rahman, Nedal Qaaud, Ashraf Emam, Nagdi M. Abdou

E39-E46

Petrological characteristics of the metamorphosed metaintrusives in amphibolite and granulite facies of the Kandalaksha part of Lapland Granulite Belt (Kola Peninsula, NW Russia) Miłosz A. Huber

Journal of Biology and Earth Sciences, Vol 3, Issue 1 , January-June 201 3

TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Influence of synthetic and plant extracts on biochemical changes in the gonadal tissue of snail Lymnaea acuminata Vatsala Singh, J.N. Tiwari Department of Zoology, TD College Jaunpur, UP, India

ABSTRACT

Fasciolosis is now recognized as an emerging human disease. Control of intermediate host is an effective tool for control of liver fluke infection. In present study effect of sublethal treatment (20% and 60% of 24h LC 50) with permethrin, cypermethrin, Allium sativum and Abrus precatorius plant extract caused a significant (p<0.05) reduction in the protein, amino acid, DNA and RNA level in the gonadal tissue of snail Lymnaea acuminata. Maximum reduction in the level of protein (33.55% of control), amino acid (1 6.21 % of control), DNA and RNA (32.96 % and 1 3.70% of control) with the treatment of 60% of 24h LC 50 of permethrin after 96h exposure periods. Among plant extract Abrus precatorius is more pronounced than Allium sativum.

Key words: Fasciolosis; Biochemical parameters; Snail; Permethrin; Cypermethrin; Plant extract.

J Biol Earth Sci 201 3; 3(2): B1 64-B1 68

Corresponding author:

J.N. Tiwari Department of Zoology, TD College Jaunpur, UP, India.

E-mail: vatsalasingh74@gmail.com Original Submission: 11 May 201 3; Revised Submission: 1 0 June 201 3; Accepted: 1 7 June 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B1 64-B1 68

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Singh and Tiwari Influence of synthetic and plant extracts on gonadal tissue of Lymnaea acuminata

INTRODUCTION Fasciolosis is an important helminth disease caused by two trematodes Fasciola hepatica and Fasciola gigantica [1 , 2]. The definitive host range of Fasciola is very broad and includes many herbivorous mammals, including humans. The fresh water snails Lymnaea acuminata and Indoplanorbis exustus are intermediate host of this parasite [3, 4].

Fasciolosis is now recognized as an emerging human disease: The World Health Organization has estimated that 2.4–1 7 million people are infected with liverfluke, and a further 1 80 million are at risk of infection [5]. Annual losses are estimated to be at least three million dollars [1 , 6]. Extensive use of synthetic molluscicides are cause serious environmental hazards. Alternatively, A number of chemically diverse plant molluscicide has been used as molluscicides as and more effective than synthetic [7]. Advantages of natural product over synthetic have eco-friendly, biodegradable abidance are less likely to accumulate in the environment.

Recently, Singh and Tiwari, [8] reported that the synthetic molluscicides and extracts of plants caused a significant reduction in the fecundity, hatchability and survival of the young snails. In the present study describe the molluscicide of synthetic and extract of plants against certain biochemical changes in the gonadal tissue of snail Lymnaea acuminata.

MATERIALS AND METHODS Test animals

Adult snails L. acuminata (2.25±0.20 cm in length) were locally collected from ponds, pools, lakes and low-lying submerged area in Jaunpur, U.P., India. The snails were acclimatized for 72 hours in dechlorinated tap water. The pH of the water was 7.1 -7.3 and dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 6.57.2, 5.2-6.3, and 1 02.0-1 05.0 mg/l, respectively.

Snails were exposed to sublethal concentrations (20% and 60% of 24h LC 50) of different synthetic (Permethrin, Cypermethrin) and Allium sativum bulb and Abrus precatorius fruit plants molluscicides. Six batches were prepared for each concentration.

Control aquarium contained only dechlorinated tap water without treatment. Different biochemical assay were performed viz. protein, amino acid, DNA and RNA in gonadal tissue of L. acuminata. After

24h the biochemical estimations were made in the gonadal tissue of the experimental snail.

Treated/control snails were removed from the glass aquaria and their gonadal tissue were dissected out, the adherent tissue was removed and the organ was put on a filter paper for absorption of water. 1 5-20 snails had to be dissected in order to obtain 1 00 mg nervous tissues.

Plants used

Plants used in this works were collected locally.

These were identified at the herbarium of the Botany Department, DDU Gorakhpur University, Gorakhpur where voucher herbarium specimens (# No 3260 for Allium sativum and # 3829 for Abrus precatorius) are on deposit.

Estimation of total protein and free amino acid

Total protein (µg/mg) was estimated according to Lowry et al., [9] using bovine serum albumin as a standard. Homogenates of gonadal tissue were prepared in 1 0% (w/v) trichloroacetic acid (TCA).

Total free amino acids (µg/mg) were determined according to the method of Spies [1 0].

Estimation of nucleic acids

DNA and RNA in gonadal tissue of L. acuminata were estimated according to Schneider [11 ] using diphenylamine and orcinol reagents, respectively.

Homogenates (1 .0 mg/ml, w/v) were prepared in 1 0% TCA at 90ºC and centrifuged at 5000 g for 20 minutes. The supernatants were used for DNA and RNA estimations.

Statistical analysis

Each experiment was replicated at least six times. Values of biochemical parameters protein, amino acids, DNA and RNA were exposing as mean ± SE. Student t-test was applied locate significance changes in between control and treated groups Sokal and Rholf [1 2].

RESULTS There was a significant (p<0.05) decrease in protein, amino acid, DNA and RNA level in the gonadal tissue of snail L. acuminata exposed to 20% and 60% of 24h LC 50 of permethrin, cypermethrin, A. sativum and A. precatorius were snail removed from 24h and 96h exposure period.

Permethrin and cypermethrin caused maximum

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Singh and Tiwari Influence of synthetic and plant extracts on gonadal tissue of Lymnaea acuminata Effect of 24h exposure to sublethal concentrations (20% and 60% of 24h LC 50) of synthetic and plants molluscicides on the level of protein (µg/mg), amino acid (µg/mg), DNA and RNA (µg/mg) in the gonadal tissue of Lymnaea acuminata.

Table 1.

Each value is mean ± SE of six replicates.

Values of parentheses are per cent change with control taken as 1 00%. *Significant (p<0.05) when t-test was applied in between treated and control groups.

Effect of 96h exposure to sublethal concentrations (20% and 60% of 24h LC 50) of synthetic and plants molluscicides on the level of protein (µg/mg), amino acid (µg/mg), DNA and RNA (µg/mg) in the gonadal tissue of Lymnaea acuminata.

Table 2.

Each value is mean ± SE of six replicates.

Values of parentheses are per cent change with control taken as 1 00%. *Significant (p<0.05) when t-test was applied in between treated and control groups.

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Singh and Tiwari Influence of synthetic and plant extracts on gonadal tissue of Lymnaea acuminata reduction of protein, amino acid, DNA and RNA level in the gonadal tissue of snail L. acuminata than the A. sativum and A. precatorius. 24h treatments with 20% and 60% of 24h LC 50 of permethrin caused maximum reduction in protein level (40.75 % and 36.59 % of control), whereas, 96h treatment caused maximum reduction in (38.04 %, 33.55 % of control) the level of protein.

Snail exposed to 20% and 60% of permethrin caused maximum reduction in total free amino acid (24h treatment -8.68 % and 7.34 % of control) and (96h treatment -6.49 % and 5.96 % of control) (Table 1 and 2). Maximum reduction 24h in the level of DNA (27.92% of control) treatment with 60% of 24h LC 50 of permethrin. Whereas, RNA level in gonadal tissue of snail L. acuminata was (1 7.41 % of control). A significant (p<0.05) decrease was observed in protein, amino acid, DNA and RNA level in the gonadal tissue of snail L. acuminata exposed to 20% and 60% of 24h LC 50 of A. precatorius after exposure 24h and 96h.

DISCUSSION It is evident from the results section that the sublethal exposure to 20% and 60% of 24h LC 50 of permethrin, cypermethrin, A. sativum and A. precatorius caused a significantly change in certain biochemical parameters in the gonadal tissue of snail L. acuminata. It indicates that the biochemical changes in the gonadal tissue are altering the reproduction behaviour of these snails.

Reduction of protein levels may be due to direct interference of the permethrin, cypermethrin and plant extract molluscicides with the protein synthesis. The synthesis of protein in any of a tissue can be affect in two ways by a chemical, (i) it either affect the RNA synthesis at the transcription stage or (ii) it some how affects the uptake of amino acid in the polypeptide chain. In this case the RNA synthesis would be inhibited resulting in reduced RNA as well as protein content and only the protein content would be affected [1 3, 1 4]. Amino acid levels in the gonadal tissue of the snail exposed to different preparation was significantly lower than control. It indicates that they also interfere with the biosynthesis of amino acid in the cell [1 5]. Singh et al., [1 4] have been reported that toxic effect of deltamethrin on the levels of biochemical changes in the snail Lymnaea acuminata. The effect of pyrethroids and fenvalerate caused a significant

inhibition in the protein level and enhancement in the amino acid level in the foot and hepatopancrease of snail L. acuminata [1 6]. The result section has been demonstrated that the following treatments with permethrin and cypermethrin in levels of DNA and RNA decrease in a time and dose dependent manner. The effect generally was more pronounced in the gonadal tissue. Thus the treatments with 60% of 24h LC 50 of permethrin and cypermethrin the both DNA and RNA levels decreased. The decrease in DNA and RNA content in L. acuminata may be because of cell death caused by release to toxic aldehydes resulting from peroxidation [1 7]. With the reduction in DNA levels change in RNA level is certainly not surprising. With the reduced RNA level there is bound to be a fall in protein levels because of reduction in the synthesis of new protein.

In conclusion, it can be stated that the component found in the synthetic (permethrin and cypermethrin) and extracts of plant alter the certain biochemical changes in snail L. acuminata.

TRANSPARENCY DECLARATION The author declares no conflicts of interest.

REFERENCES 1 . Mas-Coma S, Bargues MD, Valero MA. Fasciolosis and other plant-borne trematode zoonosis. Int J Parasitol. 2005; 35: 1 255-1 278.

2. Mas-Coma S, Valero MA, Bargues MD. Fasciola, Lymnaeids and human fascioliasis with a global overview on disease transmission, Epidemiology, evolutionary genetics. Molecular epidemiology and control. In: D. Rollinson, S. Iain Hay (Eds.). Advances in Parasitology, Burlington: Academic Press. 2009; 69: 1 41 -1 46.

3. Agarwal RA, Singh DK. Harmful gastropods and their control. Acta Hydrochim Hydrobiol. 1 988; 1 6: 11 31 38.

4. Torgerson P, Claxton J. In: Dalton JP. (Ed.), Epidemiology and Control. CAB International, Oxon.

1 999, pp. 11 3-1 49.

5. W.H.O. Report of the WHO informal meeting on use of trichlorobendazole in fasciolosis control. 1 7-1 8 October, Geneva, Switzerland, 2006. WHO. 6. Robinson MW, Dalton JP. Zoonotic helminth infections with particular emphasis on fascioliasis and trematodiases. Phil Trans R Soc. 2009; 364-376.

7. Singh A, Singh DK, Mishra TN, Agarwal RA. Molluscicide of plant origin. Biol Agric Hortic. 1 996;

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Singh and Tiwari Influence of synthetic and plant extracts on gonadal tissue of Lymnaea acuminata 1 3: 205-252.

8. Singh V, Tiwari JN. Effects of synthetic and extracts of plant on reproduction of snail Lymnaea acuminata.

Res J Pharmac Biol Chem Sci. 201 2; 3(4): 705-71 0.

9. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem. 1 951 ; 1 93: 265-275.

1 0. Spies JR. Colorimetric procedure for amino acid.

In: S.P. Colowick, N.O. Kaplan (eds.) Methods in enzymology. Acad Press, New York. 1 957, pp: 464.

11 . Schneider WC. Determination of nucleic acid in the tissue by pentose analysis. In: Colowick SP, Kaplan NO, editors. Methods in enzymology. 1 4 New York: Acad. Press 1 957; 680.

1 2. Sokal RR, Rohlf FJ. Introduction to biostatistics. WH Freeman, San Francisco. 1 973.

1 3. Tariq M, Hussain SJ, Asif M, Jahan M. Protective effect of fruit extracts of Emblica officinalis (Gaertn) Terminalia bellerica (Roxb.) in experimental myocardial necrosis in rats. Ind J Exp Biol. 1 977; 1 5: 485-486.

1 4. Singh RN, Kumar P, Singh VK, Singh DK. Toxic effect on deltamethrin on the levels of biochemical changes in the snail Lymnaea acuminata. J Pharm Res. 201 0; 3(8): 1 739-1 742.

1 5. Singh S, Singh VK, Singh DK. Effect of molluscicidal components of Abrus precatorius, Argemone mexicana and Nerium indicum on the certain biochemical parameters of Lymnaea acuminata.

Phytother Res. 1 999; 1 3: 21 0-21 3.

1 6. Singh A, Singh DK, Agarwal RA. Effect of cypermethrin mexacarbate and phorate on phospholipids and lipid peroxidation in snail Lymnaea acuminata. Bull Environ Contam Toxicol. 1 993; 51 : 68-71 .

1 7. Singh RN, Kumar P, Singh VK, Singh DK. Effect of binary combinations of deltamethrin + MGK 264 on the level of phospholipid and lipid peroxidation in the snail Lymnaea acuminata. Chemosphere. 2008; 73: 1 032-1 035.

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Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Reptilia, Squamata, Colubridae, Elaphe radiata (Boie, 1 827): New range and habitat notes for Rajaji National Park, northwest India Ritesh Joshi Conservation and Survey Division, Ministry of Environment and Forests, New Delhi, India

ABSTRACT

A new range and habitat of the copperhead snake Elaphe radiata Boie, 1 827 (Reptilia, Squamata, Colubridae) {= Coelognathus radiatus, Copper-headed Trinket Snake} has been recorded from the Rajaji National Park, north-west India. Earlier in 1 964, this species was reported from the Mohand forest located nearer to the Dehradun city, and after a gap of nearly 46 years, this documentation confirms its new range and distribution in Rajaji National Park. Two specimens were sighted randomly in Chilla and Haridwar forests of the Rajaji National Park and one in the Shyampur forest of the Haridwar forest division. All the specimens were onserved from the riparian corridors of river Ganges. I propose that this note represents the first record of genus Elaphe in the Chilla forest range of the Rajaji National Park (eastern part of the park) and in part of Haridwar forest division.

Key words: Elaphe radiata; New record; Range extension; Rajaji National Park; Northwest India.

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Corresponding author:

Ritesh Joshi Conservation and Survey Division, Ministry of Environment and Forests, CGO Complex, New Delhi – 11 0 003, India E-mail: ritesh_joshi2325@yahoo.com Original Submission: 30 April 201 3; Revised Submission: 1 0 June 201 3; Accepted: 1 4 June 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Joshi New range and habitat of Elaphe radiata in northwest India

INTRODUCTION In India, nearly 240 species of snakes occur in habitats ranging from the sea to deserts, rivers to swamps and lakes, and farmlands to mountains [1 , 2]. The Northern Regional Station of the Zoological Survey of India has compiled a consolidated document on reptilian fauna of Uttarakhand state [3], which highlighted that a total of 72 species, belonging to 46 genera, 1 4 families and 3 orders were distributed in the state. However, in sub-order Serpents, a total of 42 species of snakes were documented from the state, which belongs to five families. The Rajaji National Park (RNP), which lies in the upper Gangetic plains and the lesser Himalayan zone, has a rich diversity of flora and fauna partly characterized by a variety of habitats (presence of foothills, grasslands, Gangetic plains and different types of forests). From this protected habitat, a total of 28 snake species belonging to 11 families have been documented [4], which were based on the field observations made, records of forest department’s management plan of 1 960s–70s and information collected by locals.

The herpetofauna is one of the most poorly studied biological groups in RNP and needs

Fig. 1.

detailed studies and conservation efforts. Here, this note confirms the presence of copperhead snake Elaphe radiata in the eastern part of RNP and in Haridwar forest division (HFD). Both these habitats are same biological area having myriad types of topography and rich floral and faunal assemblage.

However, this landscape is well known for the presence of king cobra (Ophiophagus hannah) and python (Python molurus).

River Ganges is flowing across this landscape, therefore, entire area is considered as a potential habitat for herpetofaunal species. Besides, numerous seasonal water streams/annual rivers, which are tributaries to Ganges, are also flowing in between this landscape. Different habitats with varied elevations and forests further strengthen the distribution of several poisonous and non-poisonous snakes in this region.

MATERIALS AND METHODS Study area

The Rajaji National Park (Fig. 1 ; 29º5'-30º31 ' N, 77º52'-78º22' E, elevation 250–11 00 m asl) was established in 1 983 to protect Asian elephant’s habitat, which comes under ‘Shivalik Elephant

Map of the Rajaji National Park showing locations from where specimens have been documented.

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Joshi New range and habitat of Elaphe radiata in northwest India Reserve No. 11 ’. This protected habitat falls under the Gangetic plains biogeographic zone and upper Gangetic plains province. The total geographical area of the park is 820 Km 2 across the northwestern Shivalik landscape. RNP has been designated as a reserved area for the "Project Elephant" by the Ministry of Environment and Forests, Government of India with the sole aim of maintaining the viable population of Asian elephants. HFD (29º54.602’ N, 78º11 .982’ E, elevation 271 .2 m asl) is well connected with RNP and Lansdowne Forest Division and also holds a rich biological diversity, which the Rajaji carries.

The dominant vegetation of the area comprises sal Shorea robusta, rohini Mallotus phillipinensis, jhair Acacia catechu, haldu Adina cordifolia, bahera Terminalia bellirica, bar Ficus bengalensis and shisham Dalbergia sissoo. Besides, dominant fauna of the park consists of tiger Panthera tigris, leopard Panthera pardus, sloth bear Melursus ursinus, hyaena Hyaena hyaena, barking deer Muntiacus muntjak, goral Nemorhaedus goral, spotted deer Axis axis, sambar Cervous unicolor, wild boar Sus scrofa and among reptilian fauna the mugger crocodile Crocodylus palustris and king cobra Ophiophagus hannah represents Rajaji’s faunal

diverseness.

In November 201 0, after first sighting of the species and its re-sightings in April and May, 2011 , ground-based surveys were conducted on habitat type and potential sites in parts of Rajaji national Park and Haridwar forest division during day hours (09:00 h to 1 7:00 h). Besides, literature was consulted regarding its distribution and abundance in the area. Some potential riparian corridors and potential tributaries of Ganges were also walked on foot to collect more specific database on habitat types. Local people, forest officials and resource persons were also interviewed to obtain their views regarding presence of this species in and around the Rajaji National Park. Field binocular (Nikon Action series, 1 0x50 CF) was used for conducting observations and Nikon Coolpix 8700 Camera was used to capture photographic evidences. Garmin made GPS was also used to denote geographical coordinates

RESULTS AND DISCUSSION Notes on field observations

On 29th November 201 0 (1 2:1 0 h), a specimen of copperhead snake Elaphe radiata was observed

Fig. 2. Copper-head snake in the Chilla forest ofthe Rajaji National Park.

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Joshi New range and habitat of Elaphe radiata in northwest India in Chilla forest of the RNP (district: Pauri, 29º98'59" N, 78º22'80" E, elevation 299.5 m; Fig. 2). Snake was found moving slowly and inflating its head portion, particularly, its front half, intermittently while facing the people approaching the animal. After a while, more than a dozen people collected there and due to this the snake was observed frightened and trying to escape out from there. Forest officials together with local people shifted the specimen to nearby forest. Photographic evidences were collected on spot and the specimens were identified based on the literature given by various subject experts [5, 6]. Later to it, after a gap of nearly four month, on 1 3th April 2011 (1 6:47 h), I again observed a specimen of this species, crossing the Siddh Rau (annual water stream) near the Haridwar–Bijnor national highway in the HFD (district: Haridwar, 29º54'41 " N, 78º1 0'32" E, elevation 267.7 m). On 30th May 2011 , two specimens of this snake were reported from the Mayapur forest (Haridwar forest of the RNP, 29º57'97" N, 78º09'36" E, elevation 276.0 m).

The dominant vegetation recorded from the areas from where the species has been observed comprises of doob grass Cynodon dactylon, sarkanda Saccharum munja, khair Acacia catechu, shisham Dalbergia sissoo, bakli Anogeissus latifolia, gutel Trewia nudiflora, kachnar Bauhinia variegata, rohini Mallotus phillipinensis, pipal Ficus religiosa and bhang Cannabis sativa. The Shivalik range is best known for distribution of a number of torrential/annual rivers, and in monsoons when all these water channels are fulfilled with water, a number of reptiles and amphibians could be seen frequently. These water channels support the park especially in post monsoon months. Noticeably, all the specimens were observed from riparian corridors of annual rivers spread across the park, which are tributaries to river Ganges. Presence of copper-head snake in both the eastern and southwestern part of RNP indicates that this species exists in most of the ranges. However, some sites, which are closer to human settlements and annual rivers, could be considered as potential sites for its presence.

Earlier in April 1 964, this species was first reported from the Mohand forest in the Haridwar (currently part of Saharanpur district, Uttar Pradesh State) [4]. Mohand forest lies in Shivalik forest division, which is well connected with the RNP and is, situated ±40 kilometers far from the spot from

where these species have been documented. This is the first authenticated observation and evidence based documentation of copperhead snake’s presence in Chilla forest of the RNP and in the Shyampur forest of the HFD. Only seven snake species belonging to four families have been documented in the management plan of Rajaji for the period 2000-2001 to 2009-201 0 [7] and notably, this species has not mentioned in the list. However, all 28 species of Serpents have been stated in the current management plan of RNP (for the period 201 2-1 3 to 2021 -22) [8], as documented by the Zoological Survey of India [4].

Description and distribution

Copper-head snake is a large snake (up to 2.5 m in length), with a scaled copper-brown/pale yellow in colour and two distinctive transverse black stripes down the back, appearing only on half of the body. The remaining lower portion of the body is generally dark yellowish–brown in colour. The venter is yellow in colour and the eyes are relatively large. From the neck to half body portion, three dark stripes radiating from the eyes can be seen outlined in black; besides, a black stripe is also present in the neck portion, which is separating the head and body portion. When frightened, the snake inflates its fore body portion in the air (>50 cm in air) and assumes a menacing posture, which looks like S-shaped loop and an attacking posture, and is therefore, sometimes misidentified as a flying snake. Its fore body portion also become flattens when defending against threat (Fig. 3 a-c).

Copper-head snake is found across Asia; it ranges from Indonesia to Nepal, Malaysia, islands of Singapore, Myanmar, Thailand, Laos, Bangladesh and south China. In India its range was recorded from the Uttarakhand (Dehradun) to Arunachal Pradesh (Miao), parts of Madhya Pradesh (Balaghat), Chattisgarh (Bastar), Bihar (Purnia), Orissa (Cuttack), West Bengal (Darjeeling), Assam (Dibrugarh), Nagaland (Kohima) and from the Sikkim & Meghalaya states [5,6]. Recently, this species was also documented from the Ananthagiri Hills, which has highlighted its new range from the Eastern Ghats of India [9]. In 1 969, range extension of this species was noted in Doon valley and Shivaliks [1 0]. Further, in October, 2011 , this snake was rescued from Dehradun city (Rajpur area), which had confirmed its existence in Doon valley (Fig. 3 a).

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Joshi New range and habitat of Elaphe radiata in northwest India

a

b

c

(a) Defensive posture of the copper-head snake (b) Fore body portion showing scaling pattern, body colouration and compressed â&#x20AC;&#x2DC;Sâ&#x20AC;&#x2122; shape loop in first half body portion; snake generally organize its body in this shape while inflates in the air to defend, (c) head portion showing scalation pattern, eyes and tongue.

Fig. 3.

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Joshi New range and habitat of Elaphe radiata in northwest India As this species was earlier reported only from the parts of Dehradun district and western part of RNP area, which falls in the same district, this report suggest its new habitat and range in Uttarakhand state. Further based on these records from RNP, it could be concluded that the range and natural distribution of copper-head snake is extending in north-western Shivalik landscape of India. As Chilla forest lies in Pauri district of the state, there is a scope to study this species’ distribution in higher elevations (>300 m).

Copper-head snake is considered widely distributed species and considered as Lower RiskLeast Concern in Conservation Assessment and Management Plan’s workshop [11 ]. Expansion of road network in between the protected habitats and increasing rate of anthropogenic activities has been noted as the threat to the survival of this species. Recently, a study has been carried out on the road kills of wild animals in this landscape, which revealed that a total of 352 individuals of 39 species (3 amphibians, 9 reptiles, 1 8 mammals and 9 avian) have been killed on the national highways 72 (Haridwar–Bijnor), 74 (Haridwar–Dehradun) and on an ancillary road (Haridwar–Chilla–Rishikesh) in between June 2009 to May 2011 [1 2]. During the recent past, sightings of snakes in the RNP have reduced considerably; it is suspected that the spread of obnoxious weeds such as Parthenium hysterophorus ( Parthenium) and frequent forest fires have had a negative impact on the snake’s population in the forest [8].

Tropical moist deciduous forests are well known for rich floral and faunal diversity. Upper Gangetic plains province further enhanced the specie’s distribution in different ecosystems, viz. grasslands, mixed forest, sal (Shorea robusta) forests, riparian vegetation, etc. As various rivers (Ganges, Song, Suswa and Rawasan) are flowing across the RNP, diverseness of reptilian fauna could be considered unique.

Despite the fact that RNP is home to maximum reptilian species found across the Shivalik landscape, less work was carried out on the distribution and status of reptilian fauna. Therefore, there is a need to conduct field studies to review this group, which would be useful in proposing some conservation actions as well.

For effective conservation, there is also a need to coordinate ground-based surveys and to incorporate the indigenous knowledge of local

people, so that objectives related to community participation may be ensured, which are highly important in wildlife management and conservation.

In addition, photographic records/documentation together with records on GPS location of the specimen is highly required to be ensured.

CONCLUSION This study confirms the presence of the copperhead snake in RNP and HFD after a gap of nearly 46 years, since its first report. However, these observations are limited to only preliminary examinations carried out over a period of one year.

Additional detailed studies are urgently needed, which may help in strengthening our knowledge and database on the presence and distribution of Elaphe radiata and other unrecorded herpetofaunal species, especially in riparian corridors of Ganges flowing across these two protected habitats.

ACKNOWLEDGEMENTS I am thankful to the anonymous reviewers, who contributed significantly to improve the manuscript to its present form. Thanks are due to Mr. M.S. Negi and Mr. Vinod K. Shukla, RNP for providing information on specie’s presence and distribution.

Thanks are due to Dr. Abhishek Singh, Endangered Flora and Fauna on Earth Conservation Team for providing information on specie’s distribution and providing Figures 3 a-c and Mr. Alok Dixit, Doon Institute of Engineering and Technology, who has assisted in collection of field data and made dialogues with local people.

TRANSPARENCY DECLARATION The author declares no conflicts of interest.

REFERENCES 1 . Srinivasan M, Bragadeeswaran S. 2008. Reptiles. In: United Nation University’s INWEH course 1 –Training course on Mangroves and Biodiversity: pp. 438-454. http://www.ocw.unu.edu. Downloaded on 23rd May 2011 .

2. Whitaker R. 2001 . Common Indian snakes: a field guide. Macmillan India Limited, New Delhi, India.

3. Bahuguna A. 201 0. Reptilia. In: Fauna of Uttarakhand, State fauna series, 1 8 (part 1 ), Ed. Director, ZSI: pp. 445-503. Zoological Survey of

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Joshi New range and habitat of Elaphe radiata in northwest India India, Kolkata, India.

4. Husain A. Tilak R. 1 994. Snakes (Reptilia: Serpentes). In: Fauna of Rajaji National Park (Fauna of conservation area 5), Ed. Director, ZSI: pp. 91 -11 3.

Zoological Survey of India, India.

5. Smith MA. 1 943. The fauna of British India, Ceylon and Burma (including the whole of Indo-Chinese subregion), Reptilia and Amphibia, Vol.III-Serpents, Taylor and Francis, London.

6. Whitaker R, Captain, A. 2004. Snakes of India, the field guide. Draco Books, Chennai, India.

7. Pandey S. 2001 . Management plan of Rajaji National Park (2000-01 to 2009-1 0), Dehradun, Uttarakhand, India.

8. Rasaily SS. 201 2. Management Plan of Rajaji National Park (201 2-1 3 to 2021 -22), Dehradun, Uttarakhand, India.

9. Javed SMM, Tampal F, Srinivasulu C. First record of Coelognathus radiatus (Boie, 1 827) (Reptilia: Colubridae) from the Ananthagiri hills, Eastern Ghats, India. J Threatened Taxa. 201 0; 2(9): 11 72-11 74.

1 0. Bhatnagar RK. Extension of range of copper-head snake, Elaphe radiata Schlegel (Ophidia: Colubridae) to Doon valley and Doon Shiwaliks. J Bombay Nat Hist Soc. 1 969; 66(2): 383.

11 . Molur S, Walker S.1 998. Reptiles of India. In: Biodiversity Conservation Prioritization Project, India, Endangered Species Project-Conservation Assessment & Management Plan workshop: pp. 1 75.

Zoo Outreach Organization & CBSG, Coimbatore, India.

1 2. Joshi R, Dixit A. Wildlife mortality on National Highway 72 and 74 across Rajaji National Park, North India. Int J Conserv Sci. 201 2; 3(2): 1 27-1 39.

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ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Impact of salicylic acid on biofilm formation by plant pathogenic bacteria Leonid Lagonenko, Alexander Lagonenko, Anatoly Evtushenkov Department of Molecular Biology, Belarusian State University, Minsk, Belarus

ABSTRACT

The biofilm lifestyle gives bacteria distinct advantages over the planktonic lifestyle, as bacteria in such multicellular assemblages are protected from fluctuations in environmental conditions, antibiotics and host defenses. Biofilm formation, as well as motility, is controlled by density-dependent regulatory mechanism of cell-to-cell communication called quorum-sensing (QS). In this study we have shown that salicylic acid (SA), a plant-produced phenolic compound inhibits biofilm formation, motility and N-Acyl homoserine lactone production by Pectobacterium carotovorum and Pseudomonas syringae pv syringae at subinhibitory concentrations. We’ve also provided evidence that SA induces biofilm formation by Pseudomonas corrugata while inhibiting its motility. SA had no effect on biofilm formation

by Xanthomonas campestris pv campestris and slightly increased its motility. Erwinia amylovora was insensitive to SA treatment.

Key words: Salicylic acid; biofilm formation; swimming motility; AHL production; plant pathogens.

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Corresponding author:

Leonid Lagonenko Department of Molecular Biology, Belarusian State University, Kurchatova str., 1 0, 220030 Minsk, Belarus, E-mail: LagonenkoLL@gmail.com Original Submission: 02 June 201 3; Revised Submission: 07 July 201 3; Accepted: 20 July 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lagonenko et al. Salicylic acid and biofilm formation

INTRODUCTION Most bacteria commonly survive in nature by forming biofilms. W.J. Costerton described biofilms as surface-attached microbial agglomerations [1 ].

The life cycle of a biofilm includes initial attachment of bacteria to a surface, mature biofilm formation and dispersal [2]. Bacteria in such multicellular assemblages are protected from fluctuations in environmental conditions, antibiotics and host defenses by matrix composed of EPS, proteins and extracellular DNA [3]. So it’s not surprising that biofilm formation is a major virulence factor among human and plant pathogenic bacteria.

Biofilm formation is controlled by different exogenic factors. However, one of the most important factors is population density-dependent regulatory mechanism of cell-to-cell communication called quorum-sensing (QS). Such communication among Gram-negative bacteria involves N-Acyl homoserine lactones (AHLs), small secreted molecules that can be self-recognized in dosedependent manner, and a complex set of transcription factors of QS-controlled genes [4]. QS controls the expression of virulence factors among Gram-negative plant pathogenic bacteria and plays a significant role in attachment of bacteria, biofilm development and dispersal. The QS system in Gram-negative bacteria consists of LuxI-family enzyme involved in AHL synthesis and an AHL signal receptor, LuxR-family transcriptional regulator, which is activated through binding to AHL. Being activated, LuxR-like regulator induces or inhibits the expression of genes that have a sequence called “ lux-box” in their promoters. It’s notable that LuxR induces the expression of luxI thus taking part in amplifying of QS signal [5].

It has been shown that a plant-produced phenolic compound salicylic acid (SA) alters biofilm formation and motility in Pseudomonas aeruginosa, an organism causing chronic infections in patients with cystic fibrosis [6, 7]. SA is widely known as a primary plant immune response signal, but yet little is known about its effect on production of virulence factors by plant pathogenic bacteria [8]. It was reported that o-coumaric acid and t-cinnamic acid, the biosynthetic precursors of SA, induce expression of the type III secretion system genes in Dickeya dadantii and the combination of SA with t-cinnamic acid and benzoic acid induce its efflux pump gene expression [9, 1 0].

In this study we provide evidence that SA attenuates biofilm formation, swimming motility and AHL production by different plant pathogens: Erwinia amylovora (causative agent of fire blight, a disease of Rosaceae family), Pseudomonas corrugata (tomato pith necrosis), Pseudomonas syringae pv syringae (a variety of necrotic diseases of fruits), Xanthomonas campestris pv campestris (black rot of crucifers), Pectobacterium carotovorum (soft rot of fruits and vegetables). Most of these pathogens are well-studied, except of P. corrugata, which is quite a ubiquitous bacterium. Little is known about P. corrugata virulence factors, pathways of host infection and disease transmission.

MATERIALS AND METHODS In this study the following strains were used:

Erwinia amylovora 1 /79, Pseudomonas corrugata 3’M, Pseudomonas syringae pv syringae 1 3, Xanthomonas campestris pv campestris 2.5, Pectobacterium carotovorum 29 and Chromo­ bacterium violaceum CV026. All cultures were

grown on Luria Bertani (LB) agar. Overnight cultures were prepared by inoculation of 5 ml of LB liquid media and incubating at 28°C for 1 8 hours.

Salicylic acid stock solution was prepared by dissolving sodium salicylate in distilled water.

Working concentrations were 25 mM and 50 mM as the growth of bacteria at these concentrations was not affected.

Motility test

M9 minimal medium was used for motility tests [11 ]. 1 0 µl of cell suspensions in distilled water from overnight culture in LB was placed onto the plates containing M9 minimal media with 0.3% agar. The diameter of the bacterial growth halo was recorded after 48 hours of incubation at 28°С.

AHL production test

To evaluate AHL production a biosensor strain CV026 was used, as this strain produces purple pigment violacein in the presence of AHLs [1 2]. 1 0 µl of overnight culture was placed onto the surface of 0.7% LB agar mixed with 500 µl of Chromobacterium violaceum CV026 overnight culture. The diameter of the violet halo around the colony of the strain producing AHLs was measured after 24 hours of incubation at 28°С.

Chromobacterium violaceum

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In vitro crystal violet staining assay

Biofilm formation was studied using 96-well microtiter polystyrene plates [1 3]. The overnight cultures were diluted with LB to the same starting optical density. The wells of the microtiter plate were filled with 200 µl of SOBG media and then 20 µl of diluted overnight culture was added. After incubating the plate at 28°С for 24 hours, the culture medium was removed, the wells were washed once gently with distilled water and 300 µl of 1 % crystal violet solution was added to each well.

The plate was stained for 30 minutes at room temperature, the stain solution was removed and the wells were washed twice with distilled water.

Plate was allowed to dry and 300 µl of 70% ethanol solution was added to each well. The dye was allowed to solubilize by incubating the plate for 30 minutes and pipetting the content of the wells. The results were obtained by measuring OD 600.

RESULTS Recent studies have indicated that SA inhibits production of virulence factors by Pseudomonas aeruginosa [1 4]. That’s why we focused on SA in our research as this phenolic compound is found in plants and plays a significant role in plant growth,

Fig. 1.

development, transpiration and defense against pathogens [8].

In this study we used an assay proposed by O’Toole to assess biofilm formation as it’s quite simple and effective at studying factors affecting initiation of biofilm formation [1 3]. We showed that SA has strong inhibitory effect on swimming motility and biofilm formation by Pectobacterium carotovorum 29 and Pseudomonas syringae pv syringae 1 3 (fig. 1 , 2D and E). The effect was similar with data obtained earlier for Pseudomonas aeruginosa strains [6, 7]. However, it was surprising that SA inhibited swimming motility of Pseudo­ monas corrugata 3’M, while increasing its ability to form a biofilm (fig. 1 , 2C). SA had no significant impact on biofilm formation by Xanthomonas campestris 2.5 while slightly increasing its motility (fig. 1 , 2B). Erwinia amylovora 1 /79 was insensitive to SA treatment (fig. 1 , 2A).

AHL production by Pectobacterium carotovorum 29 and Pseudomonas syringae pv syringae 1 3 was also inhibited in presence of SA (fig. 3B and C).

Surprisingly, SA showed no effect on AHL production by Pseudomonas corrugata 3’M (fig. 3A).

Unfortunately, we couldn’t study the effect of SA on Erwinia amylovora 1 /79 AHL production as no violacein production by the biosensor strain was

Biofilm formation by different phytopathogenic bacteria in presence of SA.

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Fig. 2. Impact of campestris 2.5; C carotovorum 29.

SA on motility of studied strains. A – Erwinia amylovora 1 /79; B – Xanthomonas campestris pv – Pseudomonas corrugata 3’M; D – Pseudomonas syringae pv syringae 1 3; E – Pectobacterium

Fig. 3. Effect of SA on AHL production. A – Pseudomonas corrugata 3’M; B – Pseudomonas syringae pv syringae 1 3; C – Pectobacterium carotovorum 29. Violacein production was not induced by AHLs of Xanthomonas campestris pv campestris 2.5 and Erwinia amylovora 1 /79.

observed. Apparently, AHLs of Erwinia amylovora 1 /79 strains have N-acyl side chains from C1 0 to C1 4 in length and thus they are unable to induce violacein production by C. violaceum CV026, which can only be used to detect AHLs with N-acyl side chains from C4 to C8 [1 2]. The AHL production

by Xanthomonas campestris pv campestris 2.5 was not detected either, which was not remarkable as it has been shown earlier that Xanthomonas campestris lacks AHL signaling system and uses diffusible signal factors (DSF) as autoinducers instead of AHLs [1 5].

DISCUSSION Salicylic acid (SA) is an important plant hormone whose concentration in plants is regulated by abiotic and biotic factors. It’s a well-known fact that microbial infection induces accumulation of endogenous SA. As a result, the systemic acquired resistance occurs [8]. We suggested that SA not only triggers plant defense responses but also directly affects production of virulence factors by plant pathogenic bacteria.

In this study, we investigated the effect of SA on in vitro biofilm formation, motility and AHL

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Erwinia amylovora 1 /79, Pseudo­ monas corrugata 3’M, Pseudomonas syringae pv syringae 1 3, Xanthomonas campestris pv campestris 2.5, Pectobacterium carotovorum 29.

The results indicate that SA inhibits biofilm formation by Pectobacterium carotovorum 29 and Pseudomonas syringae pv syringae 1 3, induces biofilm formation by Pseudomonas corrugata 3’M. SA didn’t affect biofilm formation by Erwinia amylovora 1 /79 (fig. 1 ). SA inhibited motility of all tested strains except Xanthomonas campestris pv campestris 2.5, whose motility was slightly increased in presence of SA, and Erwinia amylovora 1 /79, whose motility was not affected

by SA treatment (fig. 2). AHL production by Pectobacterium carotovorum 29 and Pseudomonas syringae pv syringae 1 3 was inhibited by SA at 50 mM. However, SA had no impact on AHL production by Pseudomonas corrugata 3’M (fig. 3).

Plant colonization by plant pathogenic bacteria is a very complex process in which a variety of virulence factors including exopolysaccharide production and motility is involved. The previous studies have shown an inhibitory effect of SA on motility, biofilm formation and AHL production by P. aeruginosa [6, 7, 1 4, 1 6, 1 7]. These results are quite similar to those obtained for Pseudomonas syringae pv syringae 1 3 and Pectobacterium carotovorum 29.

The results above could be possibly explained by structural similarities between SA and LasR regulator ligand (LasR is a transcriptional regulator of LasI/LasR QS system in Pseudomonas aeruginosa). We may suggest that SA inhibits swimming motility and biofilm production by inhibiting the activity of LuxR-like regulator in these bacteria [5, 1 6]. The reduced ability to produce AHLs may be explained by the fact that LuxR-like regulator up regulates luxI gene expression, otherwise it remains basal. It had been shown previously that SA may also inhibit swimming motility by decreasing fliC flagellin gene expression [1 7].

However, an inhibitory effect of SA doesn’t seem to be universal. In our study, SA induced biofilm formation of Pseudomonas corrugata 3’M and didn’t show any effect on its AHL production. SA also induced motility by Xanthomonas campestris pv campestris 2.5 and didn’t affect motility and biofilm formation by Erwinia amylovora 1 /79. The absence of any effect of SA on swimming motility and biofilm

formation by Erwinia amylovora 1 /79 may be due to the presence of another signaling system in Erwinia amylovora that controls swimming motility and biofilm formation.

We may suggest that such resistance of E. amylovora, Pseudomonas corrugata and Xantho­ monas campestris pv campestris to the inhibitory effect of SA could occur as a result of coevolution of these pathogens with host plants.

Nonetheless, the results presented in this report confirm the role of SA as an antimicrobial agent that directly affects cells of plant pathogenic bacteria.

Quorum sensing, motility and biofilm formation still need to be studied to reveal all their underlying mechanisms. The various and sometimes contradictory effect of SA on these processes lets us suggest the presence of complex and overlapping regulatory networks controlling QS, motility and biofilm formation among plant pathogenic bacteria.

ACKNOWLEDGEMENTS Authors would like to thank Professor K. Geider for providing us with Erwinia amylovora 1 /79 and Dr. V. Miamin for providing us with Pseudomonas corrugata 3’M, Pectobacterium carotovorum 29, Xanthomonas campestris pv campestris 2.5.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

REFERENCES 1 . Costerton JW, Geesey GZ, Cheng KJ. How bacteria stick. Sci Am. 1 978; 238: 86-95.

2. O’Toole GA, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol. 2000; 54: 49-79.

3. Sutherland IW. Biofilm exopolysaccharides: A strong and sticky framework. Microbiology. 2001 ; 1 47: 3-9.

4. Karatan E, Watnick. P. Signals, regulatory networks, and materials that build and break bacterial biofilms.

Microbiol Mol Biol Rev. 2009; 73: 31 0-347.

5. Parsek MR, Greenberg EP. Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms. PNAS. 2000; 97: 8789-8793.

6. Chow S, Gu K, Jiang L, Nassour A. Salicylic acid affects swimming, twitching and swarming motility in Pseudomonas aeruginosa, resulting in decreased

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7. Da ML, Heroux AK, Pakzad Z, Schiffmacher KFES. Salicylic acid attenuates biofilm formation but not swarming in Pseudomonas aeruginosa. JEMI. 201 0; 1 4: 69-73.

8. Raskin I. Role of Salicylic acid in plants. Annu Rev Plant Physiol Plant Mol Biol. 1 992; 43: 439-463.

9. Ravirala RS, Barabote RD, Wheeler DM, Reverchon S, Tatum O, Malouf J, et al. Efflux pump gene expression in Erwinia chrysanthemi is induced by exposure to phenolic acids. MPMI. 2007; 20: 31 3320.

1 0. Yang S, Peng Q, San Francisco M, Wang Y, Zeng Q, Yang C-H. Type III secretion system genes of Dickeya dadantii 3937 are induced by plant phenolic acids. PLoS ONE. 2008; 3.

11 . Cesbron S, Paulin J-P, Tharaud M, Barny M-A, Brisset M-N. The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions. FEMS Microbiol Lett. 2006; 257: 221 -227.

1 2. McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, et al. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of Nacylhomoserine lactones. Microbiology. 1 997; 1 43: 3703-3711 .

1 3. Oâ&#x20AC;&#x2122;Toole GA. Microtiter dish biofilm formation assay. J Vis Exp. 2011 ; 47: 2437.

1 4. Prithiviraj B, Bais HP, Weir T, Suresh B, Najarro EH, Dayakar BV, et al. Down regulation of virulence factors of Pseudomonas aeruginosa by salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans. Infect Immun. 2005; 73(9): 531 9-5328.

1 5. He Y-W, Zhang L-H. Quorum sensing and virulence regulation in Xanthomonas campestris. FEMS Microbiol Rev. 2008; 32: 842-857.

1 6. Yang L, Rybtke MT, Jakobsen TH, Hentzer M, Bjarnsholt T, Givskov M, et al. Computer-aided identification of recognized drugs as Pseudomonas aeruginosa quorum-sensing inhibitors. Antimicrob Agents Chemother. 2009; 53: 2432-2443.

1 7. Dong Y, Huang C, Park J, Wang G. Growth inhibitory levels of salicylic acid decrease Pseudomonas aeruginosa fliC flagellin gene expression. JEMI. 201 2; 1 6: 73-78.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Indole-3-acetic acid (IAA) production by Streptomyces atrovirens isolated from rhizospheric soil in Egypt Mohamed Hemida Abd-Alla1 , El-Sayed A. El-Sayed 2, Abdel-Hamied M. Rasmey3 1Botany

and Microbiology Department, Faculty of Science, Assiut University, Egypt 2Botany Department, Faculty of Science, Zagazig University, Egypt 3Botany and Microbiology Department, Faculty of Science, Suez University, Egypt

ABSTRACT

A total of 21 0 locally actinomycetes isolates were screened for their potential to produce IAA and 1 38 (65.7%) isolates were able to utilize tryptophane and produce the IAA. The 1 2 isolates with high IAA production were secondary screened to select the most highest producer. It is worth mentioning that the most highest IAA producing isolate (22 µg/ml of IAA) was Streptomyces sp. ASU1 4 recovered from rhizospheric soil sample after wheat cultivation in New valley governorate in Egypt. TLC and HPLC analysis confirmed the IAA production in the cell filtrates of the strain. Sequencing of 1 6S rRNA genes of the Streptomyces sp. ASU1 4 isolate had 1 6S rRNA gene with 99% nucleotides identity to that of Streptomyces atrovirens NRRLB1 6357 available in Genbank database. The nucleotide sequences of the isolate Streptomyces atrovirens ASU1 4 was deposited in the GenBank nucleotide sequence database under accession number KC1 45267. IAA production by Streptomyces atrovirens ASU1 4 was optimized by studying some factors and the results revealed that the maximum IAA value was obtained when the isolate cultivated in tryptone yeast extract broth medium supplemented by tryptophane 5 mg/ml, adjusted at pH 6 and incubated at 30°C for 1 3 days. These results suggest that IAA-producing Streptomyces atrovirens ASU1 4 could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.

Key words: Indole-3-acetic acid; Streptomyces atrovirens; TLC; HPLC; 1 6S rRNA; Optimization.

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Corresponding author:

Mohamed Hemida Abd-Alla Botany and Microbiology Department, Faculty of Science, Assiut University, Egypt, E-mail: mhabdalla2002@yahoo.com Original Submission: 03 August 201 3; Revised Submission: 1 7 August 201 3; Accepted: 20 August 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B1 82-B1 93

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INTRODUCTION Little attention has been devoted on the functions of the microbial populations which impact soil processes and other life forms. Studies on the role of microorganisms and the interactions among them could allow an enhanced knowledge for a better understanding that enable life to continue [1 , 2]. The rhizosphere is an ecological niche in which develop microbial communities develop.

Many bacteria are intimately associated with plant roots. Rhizodeposition of various exudates provide an important substrate for the soil microbial community and there is a complex interplay between this community and the quantity and type of compounds released [3-5]. However, the composition and quantity of root exudates varies depending on the plant species [6] and the physical environment such as humidity and temperature [7].

There are several plant growth-promoting rhizobacteria (PGPR) inoculants currently commercialized that seem to promote growth through at least one mechanism of the following: suppression of plant disease (termed Bioprotectants), improved nutrient acquisition (Biofertilizers), or phytohormone production (Biostimulants). Commercial applications of PGPR are being tested and are frequently successful; however, a better understanding of the microbial interactions that result in plant growth increases will greatly increase the success rate of field applications [8].

Actinomycetes are Gram-positive bacteria. They are the most widely distributed group of microorganisms in nature, and are also well known as saprophytic soil inhabitants [9]. Distribution of actinomycetes has been observed in some plant rhizospheres by many authors [1 0, 11 ]. Most actinomycetes in soil belong to the genus Streptomyces [1 2, 1 3] and 60% of the sources of most biologically active compounds such as antifungal and antibacterial compounds or plant growth promoting substances that have been developed for agricultural use originated from this genus [1 4, 1 5].

The auxins are a group of indole ring compounds which have the ability to improve plant growth by stimulating cell elongation, root initiation, seed germination and seedling growth [1 6]. Indole3-acetic acid (IAA) is the main member of the auxin

family that controls many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity [1 7]. IAA also serves as a regulating agent for microbial cell differentiation, for example, it stimulates spore germination and mycelia elongation in the Streptomyces [1 8]. Several Streptomyces species, such as S. olivaceoviridis, S. rimosus, S. rochei and Streptomyces spp. from the tomato rhizosphere, have the ability to produce IAA and improve plant growth by increased seed germination, root elongation and root dry weight [1 6]. Microbial isolates recovered from the rhizosphere of different crops appear to have a greater potential to synthesize and release IAA as secondary metabolites. Production of IAA by microbial isolates varies greatly among different species and strains and depends on the availability of substrate(s). Many bacteria isolated from the rhizosphere have the capacity to synthesize IAA in vitro in the presence or absence of physiological precursors such as tryptophan [1 9]. Different bacterial pathways to synthesize IAA have been identified and a high degree of similarity between IAA biosynthesis pathways in plants and bacteria was observed [20].

Tryptophan is believed to be the primary precursor for the formation of IAA in plants and microorganisms [21 ]. However, work with tryptophan-auxotrophic mutants and isotope labeling has established that IAA biosynthesis can occur via a tryptophan independent route [22, 23], although in the presence of tryptophan, microbes release greater quantities of IAA and related compounds.

There is evidence that the growth hormones produced by the bacteria can in some instances increase growth rates and improve yields of the host plants [24]. In vivo, the effect of culture filtrates on maize and cowpea seed germination and root elongation was evaluated and compared with the commercial IAA [25]. Nowadays, some rhizosphere actinomycetes are studied and developed as a commercial product. The aim of this study was to investigate IAA production of Streptomyces sp. isolated from hizosphereic soil of some crop plants cultivated in different locations of Assiut and New Valley Governorates, Egypt and to evaluate the optimization of IAA production by an active isolate S. atrovirens ASU1 4.

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MATERIALS AND METHODS Collection of samples

A total of 1 00 rhizospheric soil samples were collected from three crop plants (Triticum vulgaris, Zea mays and Vicia faba) cultivated in different locations of Assiut and New Valley Governorates, Egypt. The samples were kept in refrigerator at 4°C until the time of isolation.

Isolation of Actinomycetes

Starch casein agar (Soluble starch 1 0.0, casein 0.3, K2HPO 4 2.0, NaCl 0.5, KNO 3 2.0, MgSO 4 0.5, CaCO 3 2.0 and agar-agar 1 5.0 g/l) and glycerol asparagine (Glycerol 20.0, L-asparagine 2.5, NaCl 1 .0, K2HPO 4 1 .0, FeSO 4•7H 2O 0.1 , MgSO 4 0.1 , CaCO 3 0.1 and agar-agar 1 5.0 g/l) with pH 7.2, supplemented with griseoflavin (50 µg/ml) and streptomycin (50 µg/ml) in order to minimize the appearance of fungi and other bacteria were used as selective media for isolation of actinomycetes.

The Streptomyces isolates were recovered by serially dilution method and the plates were rotated by hand for good dispersion of soil suspension and incubated at 28°C for 7-21 days. Individual colonies were picked up, and subcultured on fresh agar medium. The actinomycetes cultures were purified and transferred to agar slants of the same medium; incubated at 28°C for 5-7 days to achieve good sporulation, and then stored at 4°C in refrigerator.

Screening for IAA production

The production of IAA by the tested actinomycetes isolates was determined according to the method of Bano and Musarrat [26]. Discs (8 mm diameter) from colonies of the actinomycetes isolates, grown on tryptone yeast extract agar and incubated at 30°C for 5 d, were transferred to 5 ml tryptone yeast extract broth containing 2 mg/ml L-trytophan. These cultures were incubated at 30°C with shaking at 1 25 rpm for 7 d and then harvested by centrifugation at 11 ,000 xg for 1 5 min. One milliliter of the supernatant was mixed with 2 mL of Salkowski reagent [27]; the appearance of a pink color indicated IAA production. Optical density (O.D.) was read at 535 nm. The level of IAA produced was estimated against the IAA standard.

Morphological characterization of Streptomyces ASU1 4 ­ Colony characterization

The morphology of the strain was studied in tryptone yeast extract agar (ISP-1 ), oatmeal agar (ISP-3), starch agar with inorganic salts (ISP-4) and glycerol-asparagine agar (ISP-5). The production of the melanin was tested on peptone-yeast extract iron agar (ISP-6) and tyrosine agar (ISP-7). Colony morphology was recorded with respect to colour aerial mycelium, reverse side and diffusible pigment using an Inter-Society Color Council, National Bureau of Standards color chart [28]. ­ Microscopic characterization

The actinomycetes were slowly released at the intersection of medium to the cover slip. The plates were inoculated at 28±2°C for 4-8 days. The cover slips were removed from the cultured medium and observed under the light microscope. The photomicroscopy was taken using Nikon microscope.

Spore chain and spore surface ornamentation were examined by light and electron microscopy [29]. ­ Gram staining

The Gram stain was carried out on 3-4 days cultures according to the Hucker method [30]. A loopful of the freshly bacterial culture was air dried and heat fixed on a glass slide. Crystal violet stain (0.3% w/v) was added and allowed to stand for one minute. Excess stain was washed off with a gentle stream of water. Grams iodine (0.4% w/v) was added and allowed to stand for 30 seconds before being rinsed off. The stain was washed with ethanol (95.0% v/v) and then stained with the secondary stain, safranin (0.4% v/v), for one minute. This was then washed with water for 5 seconds.

Physiological and biochemical characteristics

Isomer of diaminopimelic acid was analysed

by TLC [31 ]. The physiological and biochemical characteristics were monitored according to standard methods; starch hydrolysis [32], cellulose hydrolysis [33], gelatin hydrolysis [34], casein hydrolysis [35], H 2S production [36], nitrate reduction [37] catalase production [38], milk peptonization and utilization of carbon and nitrogen sources [39].

Identification of 1 6S rRNA

Streptomyces

ASU1 4 using

­ DNA isolation

The locally isolated Streptomyces ASU1 4 was grown for 7 days on a starch agar slants at 30°C. Two ml of a spore suspension were inoculated into

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens the starch nitrate broth and incubated for 3 days on a shaker incubator at 200 rpm and 30°C to form a pellet of vegetative cells. The preparation of total genomic DNA was conducted according to Abd-Alla et al. [40]. ­ PCR amplification

The 1 6S rRNA encoding gene was amplified by the polymerase chain reaction (PCR) from purified genomic DNA using the Streptomyces – specific PCR primers StrepB, 5'- ACAAGCCCTGGAAAC GGGT-3' (forward) and StrepF, 5'-ACGTGTGCAG CCCAAGACA-3' (reverse). The PCR amplification was performed by using Qiagen Proof-Start Tag Polymerase Kit (Qiagen, Hilden, Germany). The following substrates were combined in a total volume of 25 µl including about 50 ng of template DNA, 1 2.5 µl PCR Master Mix, 5 pmol (0.5 µl) each of forward and reverse primers and the total reaction volume was completed by 11 .5 µl of water DNAase free water. This step is performed on the ice. The complete reaction mixture was incubated at automated thermocycler TC-3000 (Applied Bacteriology Laboratory, Botany and Microbiology Department, Assiut University, Assiut). The reaction conditions were: an initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 94°C for 70 s, annealing at 56°C for 40 s, and extension at 72°C for 1 30 s. A final extension was conducted at 72°C for 370 s [40]. PCR products were analyzed by electrophoresis on 1 % (w/v) agarose TBE-gels (Tris-base Boric EDTA-gel) and the gels were visualized and pictured under UV light. PCR products of about (11 00 bp) were purified from gel with the QIAquick gel extraction kit (Qiagen, Hilden, Germany). ­ DNA sequencing

Purified PCR products were sequenced by cycle sequencing with didesoxy mediated chaintermination [41 ]. Sequencing was done by the Biovision Company, which sent it to the Gene Analysis Unit (Macrogene Inc., Seuol, Korea) Sequencer AB-1 3730. For sequencing the purified PCR products, the same primers StrepB (forward) and StrepF (reverse) were used. Sequences of the 1 6S rRNA of isolates were first analyzed using the advanced BLAST search program at the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST/ in order to assess the degree of DNA similarity.

Multiple sequence alignment and molecular

phylogeny was evaluated using CLUSTALW program http://clustalw.ddbj.nig.ac.jp/top-ehtml. The phylogentic tree was displayed using the TREE VIEW program. Phylogenetic tree derived from 1 6S rRNA gene sequence was generated in comparison to 1 6S rRNA gene sequences from different standard Streptomyces strains obtained from GenBank: S. atrovirens NRRLB1 6357, S. fimbriatus DSM40942, S. heliomycini NBRC1 5899, S. cae­ lestis NRRL241 8, S. pseudogriseolus NRRLB 3288, S. cellulosae NRRLB2889, S. djakartensis NBRC 1 5409, S. carpinensis NBRC1 421 4, S. ganci­ dicus NBRC1 541 2, S. fumanus NBRC1 3042 and S. capillispiralis NBRC1 4222. ­ Nucleotide sequence accession numbers

The nucleotide sequences of the isolate ASU1 4 was deposited in the GenBank nucleotide sequence database under accession number KC1 45267.

Effect of incubation time, L-tryptophan concentration, temperature and pH on IAA production

by Streptomyces ASU1 4

The best producer isolate of IAA was chosen for further studies to optimize the environmental conditions for IAA production. The effect of incubation time was studied in tryptone yeast extract broth supplemented with 2 mg/mL L-tryptophan. Samples were drawn every 24 h for 1 5 d. The effect of L-tryptophan concentration was studied by cultivating the strain in tryptone yeast extract broth supplemented with different concentrations of L-tryptophan (0, 1 , 2, 5, 7 and 8 mg/mL). The effect of temperature and pH on IAA production was studied by cultivating the strain in tryptone yeast extract broth containing 2 mg/mL L-tryptophan at different temperatures (20-45°C) and pH levels ranging from 2.0-1 0.0 for 7 d.

Extraction, purification and detection of IAA

Streptomyces ASU1 4 was cultivated in 1 00 mL of tryptone yeast extract broth containing 2 mg/mL L-tryptophan at a pH of 7.0. IAA was extracted from the supernatants with ethyl acetate according to the method described by [42]. Ethyl acetate fractions (1 0-20 mL) were applied to TLC plates (Silica gel G f254, thickness 0.25 mm, Merck, Germany) and developed in butanone-ethyl acetate - ethanol - water (3:5:1 :1 ). Spots with Rf values identical to authentic IAA were identified under UV light (254 nm). Also, the IAA formation was

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens confirmed using UV-Visible detectors Perkin Elmer HPLC system (San Jose, California, USA).

RESULTS Screening for IAA production

A total of 21 0 actinomycetes isolates recovered from rhizospheric soil of crops cultivated in different locations of Assiut and New Valley Governorates in Egypt were screened for their ability to produce IAA. Data presented in Table 1 indicated that a total of 1 38 (65.7%) isolates have the ability to utilize tryptophane and produce the IAA but 72 (34.28%) actinomycetes isolates did not able to produce the IAA from tryptophane. The IAA producing isolates

were categorized into three groups according to the amount of IAA produced. Among 1 38 (65.7%) isolates, a total of 94, 32 and 1 2 isolates produced low concentrations (<1 µg/ml), moderate concentrations (1 -2.99 µg/ml) and high concentrations (>3 µg/ml) of IAA.

Quantitative determination of IAA by the highly producer isolates

The group of the 1 2 highest IAA producers was screened again to select the most highest producer one. Results recorded in Table 2 revealed that 4 isolates were recovered from Assiut governorate whereas the others 8 isolates were recovered from New Valley governorate. According to the cultivated

Primary screening of IAA production by actinomycetes isolates recovered from rhizosphereic soil of wheat (Triticum vulgaris), corn (Zea mays) and broad bean (Vicia faba).

Table 1.

Table 2.

Quantitative determination of IAA production by the higher producer actinomycetes isolates.

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens plant, a number of 6 isolates were from wheat, 5 from corn and one isolate from bean. It is worth mentioning that the most highest producer isolate (22 Âľg/ml of IAA) was Streptomyces ASU1 4 recovered from rhizosphereic soil sample cultivated by wheat plant in New valley governorate. This isolate was selected for further experiments.

Confirmation the IAA formation by TLC and HPLC technique

The IAA was extracted by ethyl acetate and concentrated to one ml crude extract. The crude extracts of the producer isolates were eluted on the TLC sheet against the standard IAA substance and the Rf was recorded at 0.9. Also, the IAA formation was confirmed by HPLC instrument (Fig. 1 ).

Characterization and identification of the isolate Streptomyces ASU1 4 ­ Morphological characteristics

Optical microscopic observation indicated that the aerial hyphae bear spore chains of retinaculum (RA) type (Fig. 2A). Scanning electron microscopic observation indicates that the spore was appeared oval in shape with hairy surface (Fig. 2B,C). The aerial mass colour was greenish grey, bluish green, light grey and grey on all the used media, therefore it could be assigned to the grey colour serious. The substrate mycelium colour varied depending on the medium composition and appeared as dark brown, yellow green, dark olive, olive brown, pale yellow and pale olive on the tested media. The isolate was able to produce soluble pigment of light yellowish

A) Photograph of the eluted IAA and its derivatives produced by the selected isolate Streptomyces atrovirens ASU1 4 on TLC compared with the RF value of pure IAA as standard and B) Confirmation of IAA formation in the crude extract using HPLC analysis.

Fig. 1.

Morphology of the recovered actinomycetes isolate ASU1 4. A) mycelia and spore chains under light microscope, B) and C) Scanning electron micrograph showing spore chain and spore surface.

Fig. 2.

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens Table 3.

media.

Growth patterns and mycelial colouration of the tested isolate

brown in colour. But there is no melanoid pigment produced in any tested medium by this isolate (Table 3). ­ Physiological properties

The physiological activities of the isolate Streptomyces ASU1 4 revealed that starch hydrolysis, cellulose decomposition, gelatin liquefaction, nitrate reduction, milk peptonization and catalase production were positive, but H 2S production was negative. D-fructose, D-galactose, D-xylose, sucrose and L- rhamnose were utilized for growth and no growth occurred in case of L-arabinose and lactose. Doubtful growth occurred in case of D-glucose, D-mannitol, maltose and meso-inositol. The tested isolate was able to grow on L-glutamine, L-tryptophane, L-tyrosine, L-glycine, L-asparagine, L-cystine, L-phenyl alanine, L-histidine, L-hydroxyproline, L-lysine and B-alanine. The isolate ASU31 2 was assigned to the genus Streptomyces on the basis of their morphological and biochemical characterization (Table 4).

Streptomyces

ASU1 4 on different

NRRLB1 6357 available in Genbank database. The phylogenetic tree was inferred from 1 6S rRNA sequence data by the neighbour-joining method (Fig. 3). Streptomyces atrovirens ASU1 4 on the basis of biochemical tests and 1 6S rRNA gene sequencing belongs to the family Streptomycetaceae, order Actinomycetales, class Schizomycetes.

Streptomyces atrovirens

Optimization of IAA production by Streptomyces atrovirens ASU1 4 ­ Effect of hydrogen ion concentration on IAA formation

The effect of different pH values (2, 4, 6, 7, 8 and 1 0) of the culture medium on the biomass and IAA production by Streptomyces atrovirens ASU1 4 was investigated. Both mycelial growth and IAA formation was increased gradually with the increase

­ Identification and characterization by 16S rRNA gene sequences

The phenotypic–based identification was confirmed by phylogenetic analysis. Comparison between 1 6S rRNA gene sequence of 1 054 bp of the chosen isolate ASU1 4 and 1 6S rRNA gene sequences of GenBank database as determined by using BLAST search analysis. Sequencing of 1 6S rRNA genes of the chosen isolate had 1 6S rRNA gene with 99% nucleotides identity to that of

The neighbor-joining tree based on 1 6S rRNA gene sequences showing the positions of the isolate ASU1 4 and related strains.

Fig. 3.

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Table 4. Morphological and of Streptomyces ASU1 4.

biochemical characteristics

of pH values reaching maximum at pH 6 which followed by decreasing at pH values more than pH 6 (Fig. 4). ­ Effect of incubation temperature on IAA formation

The effect of different temperatures (25, 30, 35 and 40°C) on mycelia growth and IAA production were shown in Figure [5]. The optimum temperature for IAA formation and mycelia growth by Strepto­ myces atrovirens ASU1 4 was at 30°C (Fig. 5).

­ Effect of tryptophane concentration on IAA production

IAA production by the tested isolate reached maximum amount at concentration of 5 mg tryptophane per ml of basal medium. Higher concentration of L-tryptophane above 5 g/l decreased the production both of IAA and mycelia growth (Fig. 6).

Effect of different pH values on the dry weight and IAA production by Streptomyces atrovirens ASU1 4.

Fig. 4.

Effect of temperature on the dry weight and IAA production by Streptomyces atrovirens ASU1 4.

Fig. 5.

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens

Effect of tryptophane concentrations on the dry weight and IAA production by Streptomyces atrovirens ASU1 4.

Fig. 6.

Effect of incubation periods on the dry weight and IAA production by Streptomyces atrovirens ASU1 4.

Fig. 7.

Effect of incubation period on IAA production

The mycelial growth and IAA formation by Streptomyces atrovirens ASU1 4 were estimated intervals during the incubation period of 1 4 days. It is clear that mycelial growth was increased gradually with the increase of incubation period reaching maximum after 9 days. Also, IAA formation gradually increased reaching maximum after 1 3 days of incubation (Fig. 7).

DISCUSSION Plant hormones are chemical messengers that affect a plant ability to respond to its environment.

IAA is the member of the group of phytohormone and is generally considered the most important native auxin [43]. It functions as an important signal molecule in the regulation of plant development including organogenesis, tropic responses, cellular

responses such as cell expansion, division, differentiation and gene regulation [44]. Diverse actinomycetes species possess the ability to produce the auxin phytohormone IAA, these actinomycetes produce auxins in the presence of a suitable precursor such as L-tryptophane. The two hundred and ten actinomycetes isolates which collected from 1 00 rhizospheric soil samples in this investigation were tested for their ability to produce IAA. It is worth mentioning that 1 38 isolates (65.7%) had the ability to produce IAA. Several reports have shown that different actinomycetes species from many crop rhizosphere soils have this ability [45, 46]. A total of 1 2 isolates were able to produce high concentrations of IAA ranged between 3 to 22.5 µg per one ml medium. The most active isolates were collected from the rhizospheres of wheat and corn plants. It is possible that the high levels of tryptophan will be present in root exudates of the two plants and enhance IAA biosynthesis in actinomycetes isolated from their rhizospheres.

This observation came in a good agreement with those reported by other researcher [47]. Shahab et al. [48] reported that the production of IAA by some bacterial isolates was ranged between 57-288 µg/ml. Noticeably, Streptomyces atrovirens ASU1 4 isolated from wheat rhizosphere soil showed a high ability to produce IAA and gave 22 µg/ml. No research has been reported about IAA produced by S. atrovirens isolated from rhizosphere soils. Production of IAA by Streptomyces scabies was recorded by Manulis et al. [49] and by some bacterial isolates was recorded by Meenakshi et al.

[50]. The culture filtrate of the highest active isolate Streptomyces atrovirens ASU1 4 was used to extract IAA for characterization by TLC. Chromatograms of culture extracts and standard IAA, sprayed with Salkowski's reagent, showed almost the same Rf values. The TLC findings are in agreement with other reports [51 ]. IAA production was affected by the initial pH value of the broth medium. The biomass and IAA production by Streptomyces atrovirens ASU1 4 was increased gradually with the rise of pH values until 6. Recently, it was found that the best IAA formation by Streptomyces viridis was obtained at a pH 7 [47]. Also, Yurekli et al. [52] reported that the synthesis of the highest IAA level was determined in cultures cultivated in an alkaline media at a pH 7.5. Acidic or high alkaline pH is unsuitable for IAA production because Streptomyces grow poorly in these conditions. The

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Abd-Alla et al. Indole-3-acetic acid (IAA) production by Streptomyces atrovirens pH affects the function of enzyme systems and also the solubility of many substances that are important for metabolic activities and growth of bacteria [47].

The biomass and IAA production by the tested isolate Streptomyces atrovirens ASU1 4 were investigated at different incubation temperatures from 25°C to 40°C. Streptomyces atrovirens ASU1 4 produced maximum biomass and IAA production at 30°C. Our results are consistent with the previous finding of Aldesuquy et al. [53] who recorded temperatures in the range between 25-30°C were suitable for growth and IAA production by Streptomyces sp. The maximum biomass and IAA were achieved at 5 g/l tryptophane by Streptomyces atrovirens ASU1 4. However, a higher concentration of L-tryptophane exerts an adverse effect on production. Another study indicated that Streptomyces viridis produced a high level of IAA when this isolate was grown in a medium with 2 mg/ml of L-tryptophane [47]. Similar results were also recorded by used Azotobacter spp. and Pseudomonas spp. IAA formation and the mycelial growth of the tested isolate Streptomyces atrovirens ASU1 4 were affected by the incubation period at the suitable temperature. The mycelial growth of the tested isolate reached a maximum after 9 days of incubation, whereas the maximum IAA production was obtained at 1 3 days of incubation. It has been reported that IAA production by plant growth promoting rhizobacteria can vary among different species and it is also influenced by culture condition, growth stage and substrate ability [54].

IAA can increase colonization of plant surfaces by the epiphytic an endophytic bacteria [55, 56] that enhances plant growth and yield.

CONCLUSION Streptomyces atrovirens ASU1 4 can be very effective and is potential microbial inoculant for production of IAA that can be used as plant growth promoting rhizobacteria for enriching the soil fertility and enhancing the crop yield.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

Journal of Biology and Earth Sciences

BIOLOGY

REVIEW

Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae - a review Sunil K. Singh 1 , Shailendra K. Singh 2, Ajay Singh 1 1Department

of Zoology, D.D.U. Gorakhpur University, Gorakhpur, India of Zoology, T.D. (P.G.) College, Jaunpur (U.P.), India

2Department

ABSTRACT

The medicinal plants i.e. Thevetia peruviana, Nerium indicum and Alstonia scholaris are belong to family Apocynaceae. In Ayurveda the Thevetia peruviana showed medicinal properties like bitter, cathartic, febrifuge and useful in different kinds of intermittent fevers, employed in heart diseases, asthma, and chronic diarrhoea and to stop bleeding of wounds. The Nerium indicum plant show major therapeutic activity such as analgesic, anti-ulcer and antinociceptive, antibacterial, neuroprotective, anti-diabetic, anti-oxidant activity. The Alstonia scholaris bark juice with milk has stated to be administered in leprosy and dyspepsia. Latex of this plant applied to ulcer, sores, tumours, rheumatic pain and is used for curing toothache. The biologically active compounds present in Thevetia peruviana is flavonoid, thevetin, thevetin-A, triterpenoid glycosides, Nerium indicum contains glycoside, oleandrin, tannin, volatile oil, two principle neriin and oleandrin, pseudo-akuammigine, betulin, ursolic acid, and β-sitosterol and in Alstonia scholaris are present steroids, alkaloids and triterpenoids. The present work is an extensive review of published literature concerning molluscicidal and piscicidal properties of Thevetia peruviana, Nerium indicum and Alstonia scholaris to give comprehensive information in an attempt to provide direction for further research.

Key words: Molluscicides; Snail; Lymnaea acuminata; Toxicity; Indoplanorbis exustus; Piscicidal.

J Biol Earth Sci 201 3; 3(2): B1 94-B205

Corresponding author:

Dr. Sunil Kumar Singh Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur – 273 009 (U.P), India E-mail: sunil_shrinet@rediffmail.com singhdr.sunilkumar@yahoo.com Original Submission: 1 9 July 201 3; Revised Submission: 1 7 August 201 3; Accepted: 21 August 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B1 94-B205

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae

INTRODUCTION India has one of the richest herbal medical traditions in the world. Ayurveda is the system of traditional medicine prevalent in India since 2000 B.C. Ayureveda meaning the â&#x20AC;&#x153;science of lifeâ&#x20AC;? is the oldest existing medical system recognized by WHO which is widely practiced. India is a land of immense biodiversity in which two (Eastern Himalayas and the Western Ghats of India) out of twenty five hot spots of the world are located. This country is perhaps the largest producer of medicinal herbs and is rightly called the botanical garden of the world. It is generally estimated that in India over 6000 plants are used in traditional, folk and herbal medicine, representing about 75% of the medicinal needs of the Third World Countries [1 , 2]. Medicinal herbs as potential source of therapeutic aids has attained a significant role in health system all over world for both humans animals not only in the diseased condition but also as potential material for maintaining proper health.

Interest in traditional drugs is not new but has been spurred in recent years due to an upsurge of interest in renewable resources in traditional medicine by methodological advances in pharmacology and phytochemistry. Medicinal plants are important for pharmacological research and drug development, not only when plant constituents are used directly as therapeutic agents, but also as starting materials for the synthesis of drugs or as models for pharmacologically active compounds [3].

A significant number of modern pharmaceutical drugs are thus based on or derived from medicinal plants. Traditional medicine is a powerful source of biologically active compounds. Ethnopharmacology has become a scientific backbone in the development of active therapeutics based upon traditional medicine of various ethnic groups.

The above mentioned properties of plant product have opened a new vista. In reference of India, our country possess a rich biodiversity of medicinal plants are used for many purposes. The plant products (extracts also) have been used from the immemorial. The Vrikshayurveda is the branch of Ayurveda that deals with plant health and recommends drugs possessing specific qualities of treatment of insect attack. During the co-evolution of plants and insects, plant has bio-synthesized a number of secondary metabolites to serve as defense chemicals against pest attack. Although

only 1 0,000 secondary metabolites have been chemically identified so, far the total number may exceed 400,000 [4, 5]. The many plants are belongs to family apocynaceae have been tested during past on a decade and have been shown to possess molluscicidal and piscicidal activity. Although, at present very little literature is available on the control of vector snails through plant origin pesticides, an attempt has been made in this review to assemble all the known information on molluscicidal properties of common medicinal plants of eastern Uttar Pradesh, India.

The freshwater harmful snail Lymnaea acumi­ nata and Indoplanorbis exustus is very common in the Northern part of India. Both the snails are vectors of Fasciola hepatica, which cause endemic fascioliasis in cattle and live-stock in Northern part of India [6-26]. Snail control is a somewhat neglected aspect of the control of fascioliasis.

However, it is an essential element of an integrated approach to prevent the infection caused by this parasite, which still remains one of the most important diseases in this region. The use of synthetic or petroleum based molluscicides for controlling vector snails cause serious environmental pollution [27-31 ]. To overcome the problem and to search for eco-friendly molluscicides, a number of extracts and essential oils and their isolated have been evaluated for use as molluscicides due to their high toxicity, easy availability, low mammalian toxicity, low cost and easy biodegradability [32-34]. These materials have shown encouraging results for vector controlling properties with various snail species.

Fishing with the aid of plant toxins was formerly very common. Today this easy method of fishing is still practiced in remote areas. The method is simple the poisonous ingredients are pounded and thrown in to a pool or dammed up sections of a small river.

After a short time the fish begin to raise the surface and can then readily be taken by hand. The fish can be eaten without health problems [35]. Several biocidal plants have been in use for fish catching practices by the tribal communities in large numbers including Tharu, Bhotia, Kol, Gond, Kharwar and Korwas that inhabit remote villages and forest areas of the State Uttar Pradesh and Bihar [36]. Different species of plants employed as piscicides have different effects, depending on the species of fish targeted [37]. The active principles in the plant part used (leaves, seeds, kernals and bark) have

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae varying potencies and mode of action depending on whether it is applied directly and the forms of extracts (aqueous and solvent extracts) used [38-43].

Molluscicides of plant origin Fascioliasis caused by Fasciola hepatica, the large liver-fluke, common in sheep, cattle, goat and other herbivorous animals throughout the World.

Froyed [44] reported that the 21 % cattle and 7% sheep were infected with liver-fluke in Great Britain.

In India, the freshwater snails Lymnaea acuminata and Indoplanorbis exustus are the intermediate host of Fasciola hepatica [45-52] which causes immense harm to domestic animals of this country.

Schistosomiasis is caused by Schistosoma, it is a devastating disease of mankind second only to malaria in its deleterious effect [53, 54].

Terrestrial snails and slugs cause considerable damage to both cultivated and useful non-cultivated plants. The animals can make their appearance in any damp area, snails also cause considerable damage to vegetable garden, agriculture crops and fruits orchards. Singh and Agarwal [6] reported that Pila globosa an amphibious snails causes damage to paddy crops in northern part of India. In freshwater, the larvae of parasite trematodes and thereby, cause a number of diseases. Two diseases carried by aquatic snails, schistosomiasis and fascioliasis cause immense harm to man and his domestic animals.

The best method of controlling both the diseases such as schistosomiasis and fascioliasis is chemotherapy, using orally-administered drugs for individuals with moderate or severe level of infection. The disadvantage of this approach is that it does not eliminate the infection entirely, the cost of recurrent treatment may become prohibitive and drug resistance may become a problem. A sure way to tackle the problem of schistosomiasis and fascioliasis is destroy the carrier snails and remove an essential link in the life cycle of the liver-flukes.

This can be accomplished in a number of ways including the use of many synthetic or plant molluscicides [55].

Several plants of family Apocynaceae and Euphorbiaceae have active compounds are toxic to harmful snails. Of the 1 53 crude extracts of Panamanian plants of different families, Hymenaea coubaris (Leguminosae) is most effective against

[56], Ambrosia maritime (Asteraceae) have significant molluscicidal activity against snail species Biomphalaria, Bulinu and Lymnaea, examined in field [57], sesquiterpene lactones are also responsible for the molluscicidal activity of Brazilian spp. Vernonia [58]. Shoeb et al., [59] screened many plants of family Agavaceae for their molluscicidal activity against freshwater snails. Agave attenuata powder shows effective molluscicidal activity against Biomphalaria alexandrina, Bulinus truncates and Lymnaea cailliaudi. Members of the Guttiferae are shown to contain compounds highly effective against Biomphalaria glabrata. Anacardium occidentale and Spondias mombin belongs to family Anacardiaceae shows strong molluscicidal activity against freshwater snails Biomphalaria glabrata [60].

Aqueous and alcoholic extracts of Asparagus racemosus [61 ] and Uriginia epigea leaves [62] of the Liliaceae family, exhibit high mortality rate (1 00%) against Lymnaea natalensis (LC 50 1 .0 mg/L) and Bulinus africans (LC50 50-1 00 ppm), respectively.

Pestoban (herbal molluscicide) like Cedrus deodara, Azadirachta indica and Embelia ribes have high molluscicidal activity against Lymnaea acuminata and Indoplanorbis exustus. Singh et al.

[34] have also reported that neem oil, bark, leaf and cake are potent molluscicide against Lymnaea acuminata and Indoplanorbis exustus. Singh et al., [1 9] have reported that the bark and leaf extract of Euphorbia hirta plant is highly toxic to snail Lymnaea acuminata. Yadav and Singh, [23] have also reported that the aqueous latex extracts of Jatropha gossypifolia, Euphorbia pulcherima and Croton tiglium is highly toxic to snail Lymnaea acuminata in pond conditions. Singh et al. [24] have reported that the latex extracts of Thevetia peruviana and Alstonia scholaris plants are highly toxic to target snail Lymnaea acuminata and non-target fish Labeo rohita. A list of various plants/products is belonging to family Apocynaceae which have been shown to possess molluscicidal activity against harmful snails is given in Table 1 .

Biomphalaria glabrata

Molluscicides of synthetic pesticides Godan [63], and Singh and Agarwal [46] have reviewed the various types of synthetic molluscicides available for the control of snails. The major synthetic molluscicides are metaldehyde, niclosamide, carbamate, organophosphate and

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae synthetic pyrethroids. Singh and Agarwal [46] suggested that Lymnaea acuminata which is very fast breeder throughout the year may be possibly be controlled through chemosterilization.

The molluscicidal effects of metaldehyde, have been found to depend to a great extent, on the ambient temperature and humidity. Moens [64] established the relationship between toxicity and temperature for slugs, and demonstrated that the toxicity of metaldehyde increased with a rise in temperature. The carbamate pesticides which are currently being used for the control of various species of gastropods pests are; carbaryl [65, 66], mexacarbamate and aldicarb [67] and isolan [68].

The organophosphorus compound is powerful inhibitors of cholinesterase and is therefore similar to carbamates in their mode of action. Unlike carbamate however the inhibition caused by most of these compounds is irreversible [69-71 ]. Pyrethrum (synthetic pyrethroid), which is extracted from several species of chrysanthemum plants has no lethal effect on terrestrial snails. Adlung and Kauth [72], however found that the freshwater snails, Radix auricularia, Lymnaea stagnalis and Physa fontinalis. Sahay et al. [73] studied the toxicity three synthetic pyrethroids (cypermethrin, fenvalerate and deltamethrin) singly and with the synergist piperonyl butoxide against Lymnaea acuminata and Indoplanorbis exustus. Pyrethroids are primarily on the nerve membrane by changing its permeability to Na + and K+. This causes repetitive discharges of nerves at the synapses and neuromuscular junctions [74, 75]. Singh and Agarwal [76, 77] reported that in the case of snails, permethrin, cypermethrin and fenvalerate also inhibit the respiratory enzymes, lactic dehydrogenase and cytochrome oxidases.

The studies of three pyrethroids viz. cypermethrin, permethrin and fenvalerate were tested their toxicity against the freshwater snail Indoplanorbis exustus. The LC 50 of the doses of cypermethrin is 1 .03 mg/L (24h) and 0.70 mg/l (96h); fenvalerate 1 .55 mg/L (24h) and 0.94 mg/L (96h) and permethrin 1 .7 mg/L (24h) and 0.068 mg/L (96h) against the freshwater snail Indoplanorbis exustus [78].

Piscicides of plant origin Plant extracts are referred to as botanicals and when poisonous to fish are called piscicides [79].

Such piscicidal plants contain different active ingredients known as resin, tannins, saponins, nicotine and diosgenin [80, 81 ]. However, these alkaloids are toxic to fish at high concentrations and wear off within a short time. Several plant materials have shown to be toxic to zooplanktons [82], shrimps [83] commercial fish species both in the laboratory and field studies [84, 85]. The Indian major carps Labeo rohita (Ham.) was used as the test animal because it is present in almost all freshwater reservoirs in India and is suitable for toxicity monitoring [86-88]. Recently the freshwater fish Channa punctatus was used as the test animal in almost all freshwater reservoirs in India and suitable for toxicity monitoring [43].

Different species of plants employed as piscicides have different effects, depending on the species of fish targeted [37]. The active principles in the plant part used (leaves, seeds, kernals and bark) have varying potencies and mode of action depending on whether it is applied directly and the forms of extracts (aqueous and alcohol) used [38, 39]. The toxicological actions of Thevetia peruviana, plant may be due to the presence of apigenin-5-methyl ether (flavonoid) and triterpenoid glycosides [43, 80] while pseudo-akuammigine in addition to betulin, ursolic acid and β-sitosterol, steroids and triterpenoids are present in Alstonia scholaris plant [89]. In case of karanj, Pongamia pennata seed on different fishes i.e. Gudsia giuris, Chanda nama, Oreochromis mossambicus [90, 91 ], Maesa ramentacea and Sapindus emarginatus are the most effective plants against the Moina sp.

Oreochromis niloticus and Anabas testudineus [92].

Recently, Singh et al. [43] have reported that the apigenin extracted from seed of Thevetia peruviana plant is highly toxic to fish Channa punctatus. A list of various plants/products is belonging to family Apocynaceae which have been shown to possess piscicidal activity against harmful snails is given in Table 1 .

Piscicides of synthetic pesticides During past two decades the use of pesticides has increased considerably in agriculture and in 2000, roughly around 5400 million pounds of pesticides were applied throughout the world, of which 23% was accounted by US alone [93]. The pollution of rivers and streams with chemical contaminants has become one of the most critical of

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae the pollutants transport from industrial areas into the environment and their chemical persistence, many freshwater ecosystems are faced with spatially or temporally alarming high levels of xenobiotic chemicals [94, 95]. Contamination of aquatic ecosystem by pesticide can cause acute and chronic poisoning of fish and other organism. The pesticides are found to damage vital organs of fish [96]. Malathion is introduced into the environment; it may cause serious intimidation to the aquatic organisms and is notorious to cause severe metabolic disturbances in non-target species like fish and freshwater mussels [97, 98]. Malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate), a commonly used organophosphate, is applied for mosquito control [99].

Ethnopharmacology Ayurveda describes the plant as bitter, cathartic, febrifuge and useful in different kinds of intermittent Table 1. A list

fevers, employed in heart diseases, asthma, and chronic diarrhoea and to stop bleeding of wounds.

The Alstonia scholaris fresh bark juice with milk has stated to be administered in leprosy and dyspepsia.

Latex of this plant applied to ulcer, sores, and tumours and in rheumatic pain and is used for curing toothache. Thevetia peruviana and Alstonia scholaris, are common medicinal plants of India.

The latex of Thevetia peruviana is used in teeth cavities for relief from toothache and the latex of Alstonia scholaris is applied to ulcer, sores, tumors and rheumatic pain and is used for curing toothache [1 00]. The Nerium indicum plant show major therapeutic activity such as analgesic, anti-ulcer and antinociceptive, antibacterial, neuroprotective, anti-diabetic, anti-oxidant activity. All the plants have potent molluscicidal and anti-cholinesterase activity against harmful snails and slugs [9, 1 3, 1 7, 1 01 ].

The toxicological actions of Thevetia peruviana, may be due to the presence of apigenin-5-methyl ether (flavonoid) and triterpenoid glycosides [80]

of genera in the plants family Apocynaceae with molluscicidal and piscicidal activity.

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae while a number of alkaloids (pseudo-akuammigine in addition to betulin, ursolic acid and β-sitosterol), steroids and triterpenoids are present in Alstonia scholaris [89, 1 02]. The Nerium indicum plant contain glycoside, oleandrin, tannin, volatile oil, 0.25% leaves two principle neriin and oleandrin, glucosides with properties similar to digotalin the main toxin of oleander is oleandrin [1 03, 1 04].

Herbal uses of India in the form of medicine, natural pesticide, allelopathic herbs etc. emphasize that it is a plant of high potential.

Pharmacology Thevetia peruviana

The toxicological actions of

Table 2.

Thevetia peruviana

may be due to the presence of apigenin-5-methyl ether (flavonoid) and several other triterpenoid, glycosides etc. [80]. The latex, bark and leaf of Thevetia peruviana plant is used for control of harmful snails in this region [8, 9, 1 3, 1 5, 1 8, 26].

Recently, Singh et al. [24, 43] have reported that the latex extract of Thevetia peruviana plant is highly toxic to target snail Lymnaea acuminata and nontarget fish Channa punctatus and Labeo rohita.

Nerium indicum

This plant contain glycoside, oleandrin, tannin, volatile oil, 0.25% leaves two principle neriin and oleandrin, glucosides with properties similar to digotalin the main toxin of oleander is oleandrin [1 03, 1 04]. The bark of Nerium indicum plant is

Comparison of LC 50 to plant origin pesticides and synthetic pesticides against snails.

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Singh et al. Molluscicidal and piscicidal properties of three medicinal plants of family Apocynaceae Table 3. Comparison

of LC 50 to plant origin pesticides and synthetic pesticides against fishes.

used for control of harmful snails [48].

Alstonia scholaris

The pseudo-akuammigine in addition to betulin, ursolic acid and β-sitosterol, steroids and triterpenoids are present in Alstonia scholaris [89, 1 02].

The latex, bark and leaf of Alstonia scholaris plant is used for control the harmful snails [8, 9, 1 3, 1 5, 1 7, 1 8, 24, 26].

CONCLUSIONS In the present review summarizes the updated information concerning the molluscicidal and piscicidal claims and scientific studies of family apocynaceae plants. All plants have immense potential and have broad spectrum of activity on several aliments. Various parts of all the plants are used as bitter, cathartic, febrifuge and useful in different kinds of intermittent fevers, employed in heart diseases, asthma, and chronic diarrhoea and

to stop bleeding of wounds. The Nerium indicum plant showed medicinal properties like analgesic, anti-ulcer and antinociceptive, antibacterial, neuroprotective, anti-diabetic, anti-oxidant activity. The Alstonia scholaris fresh bark juice with milk has stated to be administered in leprosy and dyspepsia.

Latex of this plant applied to ulcer, sores, and tumours and in rheumatic pain and is used for curing toothache. It is reported to contain apigenin5-methyl ether (flavonoid), thevetin apigenin, several other triterpenoid, glycosides, (pseudoakuammigine in addition to betulin, ursolic acid and β-sitosterol), steroids and triterpenoids in different parts of the all the plants. Review of literature also pin points that although number of diseases for which Thevetia peruviana, Nerium indicum and Alstonia scholaris finds its uses in traditional system is fairly large, though its therapeutic efficacy has been assessed in few possible medicinal application.

There are a very large number of plants, which

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After all such compounds are not only confined to the plants in which they are founds but also possibility gets distributed in the environment. We strongly feel that if these herbaceous products are used as molluscicides they would not only control the vector snail, predatory fish and mosquitoes but would also have the advantage of easy availability, low cost biodegradability and greater acceptance amongst the users. Furthermore, we feel that with further progress in biotechnology, such products could be raised form, sources other than those plants in which they are currently found. Production of plant pesticides could, in long run also become an important industry using biotechnological methods.

While there is much information on the toxicity and lethal doses of these plant pesticides, very little literature available on their mode of action and effect on other organism. We are hopeful that pesticides of plant origin will find practical use as many of the plants having molluscicidal and piscicidal activity have been use by for Integrated Pest Management Programme (IPM) and safe environment for human beings.

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TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

The effects of soil heavy metals pollution and seasonal variations on gametogenesis and energy reserves of the land snail Eobania vermiculata Abdelmonem Mohamed Khalil Department of Zoology, Faculty of Science, Zagazig University, Egypt

ABSTRACT

Various substances like pesticides, industrial chemicals and some natural products have the potential to alter hormonal pathways that regulate the reproductive process in certain animal species. In the present study, reproductive cycle of the land snail, Eobania vermiculata from two different Governorates in Egypt has been investigated. Biochemical analyses of the mantle and gonad confirmed an annual cycle with a winter and an autumn inactive period followed by a rapid gonad development in spring and summer.

This study has shown that the condition factor (CF) and the Gonado-somatic index (GSI) values were significantly higher (P < 0.05) in snails collected from Abu-Zabaal village (TR) than that collected from New Salheia city (NS). Pattern is evident for lipid and glycogen levels in the gonad which reflects the vitellogenic process. The results of the present study can distinguish snails collected at TR according to a belated gametogenesis concomitant with a significant higher gonad glycogen concentration observed in August and September. A persistent dysfunction of a vitellogenic process is suspected to be due to the exposure to anti-estrogenic contaminants.

Key words: Soil pollution; Gametogenesis; Condition factor; Gonado-somatic index; Energy reserves; Eobania vermiculata.

J Biol Earth Sci 201 3; 3(2): B206-B21 3

Corresponding author:

Abdelmonem Mohamed Khalil Department of Zoology, Faculty of Science, Zagazig University, Egypt E-mail: abdulmonem111 @gmail.com Original Submission: 05 August 201 3; Revised Submission: 1 0 September 201 3; Accepted: 11 September 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B206-B21 3

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INTRODUCTION The terrestrial environment receives a wide range of chemicals from natural and anthropogenic contaminant inputs. In the last 50 years, large number of factories have been built in Egypt. The released contaminants are negatively affecting on the society and national economy. Interest in achieving standard rates of industrial development without previous environmental planning resulted in natural resources deterioration and increased losses and consumption of raw materials. This has basically contributed to ambient environment (water, air, and soil) pollution [1 ]. In the last few decades, land gastropods have greatly increased in economic importance and they are considered group of the most serious pests attacking agricultural crops around the world. They cause considerable damage to field crops, vegetables, fruit trees and ornamental plants. The brown garden snail, Eobania vermi­ culata (Mßller 1 774), is one of the most dangerous land snail species that has been recorded in Qalyubiya and Sharkia Governorates by many authors [2-11 ]. E. vermiculata has been used as a standard snail species for in vivo and in vitro bioassays and was supported as a useful bioindicator organism for terrestrial pollution biomonitoring studies [1 2]. Energy reserves are considered as biomarkers to reflect sublethal changes resulting from a stressful xenobiotic exposure [1 3]. The range of the energy depletion would reflect the strength of the stress [1 4-1 6] but seasonal energy storage and utilization in land snail molluscs are closely linked to environmental conditions [1 7-1 8]. Reproduction as well as gametogenesis, is controlled by a neuroendocrine system, which is dependent on external stimuli [1 9-21 ]. The objectives of this study were to determine the effects of unpolluted and polluted soils on the sexual maturation and gametogenesis of land snail E. vermiculata and also to determine the effects of seasonal variations on the energy reserves that are used in gonad development.

MATERIALS AND METHODS Sampling and site selection Adult snails (shell height 28 Âą 2.1 mm) were collected monthly from April to September 201 2 at two sampling sites in Qalyubiya and Sharkia Governorates. The first site, Abu-Zaabal (TR), is

located in the Northeast of Cairo at the point 1 8 km on Cairo-Ismailia road (Agricultural Road) with a total area of 56 km. TR was built as residential, industrial, and agriculture village. It has been submitted to several pollution sources generated by municipal sewer and waste of factories. AbuZaabal phosphate fertilizer factory is the principal source of pollution in this region. The second site, New Salheia (NS) is located in the Southeast of Sharkia Governorate. It is newly agricultural reclaimed area (1 000 acres) and depends on ground water for drinking and irrigation. This site is not submitted to any potential input of contaminants and will help to explain the influence of particular environmental conditions on the physiology and the reproductive status of snails. Five soil sub-samples (50 g/sub-sample) and five snail sub-samples (1 0 snails/sub-sample) were collected from field crops of each site. Snails were immediately preserved with ice after sampling process. Upon arrival to the laboratory of invertebrates in Zagazig University, morphometrics were assessed to each tested snail. Length was measured to the nearest 0.1 mm using digital caliper. Total fresh tissue weight and wet gonad weight were weighed by digital balance with 0.1 mg accuracy.

Physical-chemical soil analyses The alluvial soils in Egypt are located in the Nile valley and delta (Qalyubiya and Sharkia Governorates), and are the direct result of the Nile River's seasonal flooding. Alluvial soils that are formed by river sediment deposits are called fluvial.

The alluvial fluvial soil of the Nile valley is finegrained and extremely fertile, because it is composed of natural minerals and organic material.

Physical-chemical soil analyses were performed in triplicate, after mixing three of the 5 soil-subsamples for each site to obtain homogeneous samples. The soils were characterized for pH, organic matter content (OM), and water holding capacity (WHC) following the methods of Allen [22]. Heavy metal concentrations were assessed following the methods of Santorufo et al. [23].

Condition factor and Gonado-somatic index Condition factor (CF) and Gonado-somatic index (GSI) have been calculated using the following equations [24-25]:

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Biochemical analyses

Mean ± SD pH, organic matter content (OM), Water holding capacity (WHC) and metals concentrations (Cd, Cu, Pb, Zn) of soils at TR and NS sites.

Table 1.

Little pieces of the mantle and the gonad were homogenized in 0.1 M phosphate buffer, pH 7.0 for proteins and lipids analyses or 0.1 M citrate buffer, pH 5 for glycogen analysis and conserved at -4 ºC. Total proteins were determined in the gonad and the mantle according to the protein - Coomassie blue dye binding principle [26] using bovine serum albumin as the standard. The phosphovanillin method [27] was used for lipid determination and glycogen was estimated after an enzymatic digestion of the gonad and mantle homogenates with amyloglucosidase according to Carr and Neff [28]. Olive oil and glycogen were used as standards for each method, respectively.

Statistical analysis Results from each site were compared from April to September 201 2. All comparisons for statistical significances were made using a one-way analysis of variance to examine differences between the means of each biochemical biomarker. A t-test was performed for single comparisons in the presence of a parametric distribution of the values. Microsoft Excel 201 0 has been used to draw the graphs and the statistical software, Statview5, has been used to statistically analyze the data.

RESULTS Soil physical and chemical properties Soil of NS showed pH around neutrality (7.11 ± 1 .2) while it was slightly acidic (5.4 ± 0.5) for soil of TR (Table 1 ). Percentages of OM and WHC were particularly high at NS (1 9.6 ± 2.6 and 33.5 ± 3.8, respectively) (Table 1 ). With the exception of Cd, the concentrations of all examined heavy metals were nearly equal at both sites. Cd concentration at TR (1 26.6 ± 1 0.5 mg kg -1 soil dry weight) was extremely higher than that at NS (2.5 ± 0.4 mg kg -1 soil dry weight) (Table 1 ).

Condition factors and gonado-somatic index Seasonal variations in condition factors (CFs) of

Condition factor (CF) values (mean ± SD) from April to September 201 2 of E. vermiculata at Ns and TR sites.

Fig. 1.

snails collected from the two sampling sites are represented in Fig. 1 . CF patterns of snails collected from TR were different than that of snails collected from NS. In September, CF has never exceeded 5% at TR whereas it has never fallen under 5% at NS. CFs of TR snails were statistically smaller (P < 0.05) than that of NS snails during May, July, August and September. Generally, CFs did not significantly vary for snails that were sampled in the TR. Variations of gonado-somatic index (GSI) (Fig. 2) showed, as previously observed with CF, two different distribution patterns according to the sampling site location. At NS sampling site, GSIs remained nearly constant over the season (from June to September) and were significantly higher (P < 0.05) than GSIs determined in the TR from July to September (between 1 4.9 and 1 4.1 5%). In the NS, GSI reached a peak (1 6.32%) in May, but it took 1 month more for TR snails to reach a peak (1 2.2%) in June. GSIs were significantly (P< 0.05) higher for NS snails (1 6.32%,

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Gonado-somatic index (GSI) values (mean Âą SD) from April to Semptember 201 2 of E. vermiculata collected at NS and TR sites.

Fig. 2.

1 4.9%, 1 4.87% and 1 4.1 5%) than that of TR snails (9.78%, 9.11 %, 8.69% and 8.41 %) in, May, July, August and September, respectively.

Energy reserves Seasonal variations of protein, glycogen and lipid concentrations in the mantle and the gonad are represented in Figs. 3 & 4 for snails of NS and TR, respectively. In NS and in April, May and June (Fig. 3), protein concentrations were significantly higher in the mantle (77.81 , 93.49 and 49.67 mg g -1 wet wt., respectively) than that in the gonad (53.59, 47.57 and 28.44 mg g -1 wet wt., respectively) (P< 0.05) but they were significantly lower (P< 0.05) in July, August and September (38.51 , 27.1 6 and 23.82 mg g -1 wet wt. respectively) than that in the gonad (77.43, 63.42 and 34.66 mg g -1 wet wt. respectively). Highest protein concentrations were observed in May in the mantle (93.49 mg g -1 wet wt.) and in July in the gonad (77.43 mg g -1 wet wt.).

In the latter, the lowest protein concentration was observed in June (28.44 mg g -1 wet wt.) whereas a significant increase (P< 0.05) could be noticed between June and July (28.44 and 77.43 mg g -1 wet wt., respectively). Lowest glycogen concentrations were found in April and May in the gonad (5.45 and 2.84 mg g -1 wet wt., respectively) and then a significant increase (P < 0.05) was observed from June to July (from 1 0.28 to 32.63 mg g -1 wet wt.).

Such a pattern was not observed in the mantleâ&#x20AC;&#x2122;s glycogen in which concentrations remained higher (P < 0.05) than those from the gonad from April to June (between 1 3.44 and 20.22 mg g -1 wet wt.). In May, lipid concentrations in the gonad were

Concentrations of protein, glycogen and lipid (mg g -1 wet wt.) in gonad and mantle of snails collected at NS site from April to September 201 2.

Fig. 3.

significantly (P <0.05) lower than that recorded in the mantle (7.1 8 and 22.23 mg g -1 wet wt. respectively) while these concentrations significantly increased to be 39.69 and 35.38 mg g -1 wet wt. in August and September, respectively. Highest lipid concentrations could be seen in July in mantle (33.01 mg g -1 wet wt.) and in gonad (42.81 mg g -1 wet wt.). The same seasonal pattern could also be observed with lipid and glycogen concentrations in the gonads of TR snails (Fig. 4). Lipid concen-

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Khalil The effects of soil heavy metals pollution on snail Eobania vermiculata wet wt., respectively) were higher (P < 0.05) in the gonad of TR snails than that of NS snails. Snails from TR had also higher lipid concentrations in the gonad than snails from the other site from April to May (23.9 and 1 6.25 mg g -1 wet wt., respectively).

DISCUSSION

Concentrations of protein, glycogen and lipid (mg g -1 wet wt.) in gonad and mantle of snails collected At TR site from April to September 201 2.

Fig. 4.

trations decreased in the gonad from April to June and increased in July (P <0.05) until September. In TR, glycogen concentrations also remained low in the gonad from April to June but increased and became significantly higher (P <0.05) than that in the mantle from July to September. Gonads of TR snails had higher (P< 0.05) lipid concentrations in April (23.9 mg g -1 wet wt.) than that of NS snails (1 2.4 mg/g -1 wet wt.) but from June to August, lipid and glycogen concentrations (between 1 9.29 and 39.69 mg lipids g -1 wet wt. and between 1 0, 28 and 35.1 6 mg glycogen g -1 wet wt., respectively) were higher (P< 0.05) in gonad of NS snails. In April, lipid and protein concentrations (23.9 and 64.48 mg g -1

Temperate-zone snails such as E. vermiculata (present study), Helix pomatia and Helix aspersa [29-30] have both clear hibernation periods with no reproductive activity during the year (autumn and winter) and a breeding period. Otherwise, tropical species such as Gyliotrachela depressispira [31 ] show continuous spermatogenesis during the year while oocyte diameters, female reproductive system maturation and oviposition are strictly correlated with periods having increased precipitation [32].

Throughout the reproductive period, sexual maturation and the energy status of land snails are likely to respond to numerous environmental conditions such as food abundance and contamination levels [33-37]. Environmental conditions in Sharkia Governorate are characterized by a spatial heterogeneity of abiotic factors [38]. Snails from NS had all along the season, higher CFs than snails from TR. Values of the GSI also reflect very well these differences in support of nutritive conditions in both environments: higher and nearly constant GSI from July to September for snails from NS whereas a single and little peak appears during the reproductive period of snails from TR. Snails from NS have shown a reproductive period earlier in the spring and a second maturation period at the end of summer and the beginning of autumn. In comparison, the delayed, single and short reproductive period observed for snails from TR might reflect the limited nutritive conditions and the abundance contaminants in this area.

The relationship between gametogenesis and energy metabolism is highly consistent from May to September. Lowest glycogen concentrations were observed in April and May in gonad of snails from NS. In addition to gonad development, significant increase of lipid concentration was effective from July to September. Higher lipid levels were also found in the ovotestis of Megalobulimus oblongus in winter and spring [1 7]. Lipids are used for the production of gametes in the pulmonate Semperula maculata [39]. Despite a delayed sexual maturation for snails collected in TR, the same seasonal

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Khalil The effects of soil heavy metals pollution on snail Eobania vermiculata pattern is effective for gonad glycogen levels, and the lowest lipid concentrations were also observed in July during the oviposition period. An increment in the vitellogenic process due to the action of the dorsal body hormone [1 7] could explain the sudden increases in the mean GSI observed from winter to spring. The reduction of the GSI obtained in the summer compared to the spring is probably due to the ovulation or degeneration of a great number of ripe oocytes at the end of spring and beginning of summer, and to a depression of the vitellogenetic process. Spring, is a period characterized by pronounced copulation and egg laying in several species of snails and slugs dwelling in places having temperate or tropical climates [40-46]. When Helix aspersa was submitted to a decreased light regime in a 24-hours light/dark cycle or a fixed short day (8 hours light: 1 6 hours dark), it presented a partial or complete reduction in reproductive activities such as mating, egg-laying, and ovulation [47]. The results of the GSI suggest that spring is the principal reproductive season in the annual reproductive cycle of E. vermiculata. Increases in the light/dark ratio and the successive temperature rises that characterize spring have a direct influence on the reproductive activity of other Pulmonates such as Helix aspersa [48], stimulating mating and egg laying [49]. Organic contaminants (especially, Polycyclic aromatic hydrocarbons) like metals and especially Cd could be suspected to act singly or in synergy as endocrine disruptor on the metabolism controlled by estrogens or on estrogen metabolism itself through cyt. P450 induction [50]. Beside these observations, the biochemical results of the present study reveal other perturbation effects on snails from TR involved by a contaminant exposure. In May, their lipidic content in the gonad was significantly higher in comparison with snails from NS despite a significant delayed sexual maturation, and in June, lipidic content of the mantle was very depressed. These results might be indicative of a contaminant exposure on energetic metabolism of snails from TR. Energy costs induced by contaminant exposure is well documented for molluscs and many disturbances in the reserves occur in certain tissues due to a dysfunction of physiological process controlling numerous pathway of catabolism, metabolism and liberation of glycogen or lipids [51 -52].

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Vegetation analysis and species diversity in the desert ecosystem of coastal wadis of South Sinai, Egypt Fawzy Mahmoud Salama1 , Monier Mohamed Abd El-Ghani 2, Salah Mohamed El-Naggar1 , Mohamed Meftah Aljarroushi 3 1Botany

Department, Faculty of Science, Assiut University, Assiut, Egypt Department, Faculty of Science, Cairo University, Egypt 3Botany Department, Faculty of Science, Muserata University, Libya 2Botany

ABSTRACT

This study aims to investigate the floristic composition, biological spectrum, chorological affinities, and describes the vegetation inhabiting the main channel and the deltaic part of Wadi Kid as one of the principal coastal wadis in South Sinai. The life-form spectrum in the present study is characteristic of an arid desert region with the dominance of therophytes (30.43% of the total) and chamaephtyes (26.09%), followed by hemicryptophytes (26.09%), phanerophytes (1 4.49%), geophytes (1 .45%) and parasites (1 .45%). Phytogeographically, the Saharo-Arabian element forms the major component of the floristic structure. The investigation revealed that Wadi Kid is potential shelters of 5 vegetation groups.

Detrended Correspondence Analysis (DCA) represented the distribution of the recognized groups along the first two axes. Canonical Correspondence Analysis (CCA) indicated that clay, coarse sand, electric conductivity, chlorides, magnesium and calcium were the main soil parameters which determined the distribution of vegetation in the study area.

Key words: Wadi Kid; Floristic analysis; Coastal wadis; Soil-vegetation relationships; Multivariate analysis.

J Biol Earth Sci 201 3; 3(2): B21 4-B227

Corresponding author:

Fawzy Mahmoud Salama Department of Botany, Faculty of Science, Assiut University, Assiut, Egypt E-mail: fawzy_salama201 0@yahoo.com Original Submission: 09 July 201 3; Revised Submission: 1 9 September 201 3; Accepted: 23 September 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B21 4-B227

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt

INTRODUCTION Southern Sinai is triangular mass of mountains, about 28400 km 2 [1 ] in surface area, of igneous and metamorphic rocks. This mass of mountains is intensively rugged and dissected by a complicated system of deep wadis, some of which reach a considerable length (e.g. Wadi Feiran, and Wadi Zaghar) and some are shorter, narrow and steeper, and represent tributaries of the main wadis (e.g.

Wadi El-Arbaein) [1 , 2]. This roughness in geomorphology lead to differentiation of enormous number of microhabitats [3] and landforms [2] which resulted in relatively high diversity in ecosystems and flora. This area is intensively rugged and dissected by a complicated system of flash floods, but under normal circumstances most of the water is underground, occasionally surfacing to produce short sections of freely flowing permanent water.

Sparse vegetation occurs everywhere, but the wet areas are particularly rich with plants and consequently with insects and other animals. The plant life in Sinai has proved a very interesting topic for many botanists and explorers over the years [4-6]. The Plant Red Data Book of Egypt shows that 1 42 species of trees and shrubs were already threatened by the early 1 990 [7, 8].

Wadi Kid is a main basin which drains to the Gulf of Aqaba [1 ]. It is a long wadi, located about 50 km north to Sharm El-Sheikh city, and extends for about 50 km, surrounded by different types of granitic and volcanic mountains. The width of the main wadi ranges between 50-1 00 m, and in some areas turned into vast plains. This wadi can be divided into two main parts, the upstream part and the downstream part. The upstream part is mainly gravel with surface cobbles. The downstream part of the wadi is covered mainly with rocks (cobbles and stones) with a coarse sand strips near the foot hills.

The present study aims to investigate the floristic composition, biological spectrum, chorological affinities and describes the vegetation inhabiting the principal channel of Wadi Kid including its deltaic part using multivariate analyses technique.

and extends for about 50 km long with an elevation ranged between 1 7 and 636 m a.s.l. Wadi Kid is easily traversable, in most parts, by appropriately equipped vehicle. In some locations of rough rocky terrain, needing attention for its vegetation, were reached on foot. The study was carried out along two successive years 201 0 and 2011 . Forty georeferenced stands in the deltaic part and along the main trunk of the wadi were studied (Fig. 2). These stands studied were randomly chosen at locations where considerable vegetation cover was encountered.

The Raunkiaer system [9] was used to classify the recorded species. The number of species within each life form was expressed as a percentage of the total number of species. Species richness (SR) within each separated vegetation group was calculated as the average number of species per stand. The Shannon-Wiener diversity index was calculated from the formula H′=-ΣPi lnPi [1 0], where, H′ is Shannon-Wiener diversity index and Pi

Fig. 1.

Location map of Wadi Kid.

Fig. 2.

Location of studied stands inside Wadi Kid.

MATERIALS AND METHODS Wadi Kid is the longest wadi after Wadi Feiran in South Sinai (Fig. 1 ). It is located between longitude (34°09`& 34°27`E) and latitude (28°04`& 28°21 `N),

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt is the relative presence value of the ith species.

Materials about local flora [11 -1 6] were consulted for taxonomic nomenclature. The phytogeographical range of species distribution was carried out according to Zohary [1 7] Abd El-Ghani [1 8] and Hassan [1 9]. Voucher specimens were deposited at the Herbarium of the Botany Department, Faculty of Science, Assiut University. Duplicate series of specimens were also deposited at the Herbarium of Cairo University.

For each sampled stand, three soil samples were collected from profiles of 0â&#x20AC;&#x201C;50 cm depth.

These samples were then pooled together to form one composite sample, air-dried and thoroughly mixed. Textures were determined by sieving method to separate gravels, coarse sand, fine sand, silt and clay. Determination of electric conductivity and pH was determined in soil-water (1 :5) extracts. Calcium and magnesium were determined volumetrically by the versene titration method described by Johnson and Ulrich [20]. Sodium and potassium were determined by flame photometry according to Williams and Twine [21 ]. Estimation of chlorides was carried out by titration methods using 0.005N Silver Nitrate [22, 23]. Sulphates were determined according to Bardsley and Lancaster [24].

A floristic data matrix of 40 stands and 69 species was subjected for classification by cluster analysis of the computer program MVSP version 3.1 [25] using squared Euclidean distance dissimilarity matrix with minimum variance (also called Wardâ&#x20AC;&#x2122;s method) as agglomeration criterion [26]. The computer program CANOCO version 4.5 [27] was used for all ordination analyses whereas the computer program SPSS version 1 0.0 [28] was used for all the statistical treatments. Principal Component Analysis (PCA) was used to identify the main gradients that influence species distribution.

Canonical Correspondence Analysis (CCA) was the appropriate ordination method to perform direct gradient analysis [27]. Due to high inflation factor of pH, it was excluded from analysis. Therefore, CCA was performed using 1 3 environmental variables: gravels, coarse sand, fine sand, silt, clay, electric conductivity (EC), total soluble salts (TSS), sodium, potassium, calcium, magnesium, chlorides and sulphates. All the default settings were used for CCA, and a Monte Carlo permutation test (499 permutations [29] ) was used to test for significance of the eigenvalues of the first canonical axis.

RESULTS Floristic analysis

Altogether 69 species (20 annuals and 49 perennials) belonging to 57 genera in 33 families were recorded. The largest families were Asteraceae and Zygophyllaceae (8 and 7 species, respectively), Caryophyllaceae, Boraginaceae, Brassicaceae, and Fabaceae (4 species for each) and Geraniaceae, Resedaceae, Poaceae (3 species for each). They constituted most of the floristic structure in South Sinai Desert, whereas Asclepiadaceae, Chenopodiaceae, Cleomaceae, Cucurbitaceae and Polygonaceae were equally represented (2 species for each). Nineteen families were represented by only one species. The largest genera were Fagonia and Zygophyllum, 3 species for each (Table 1 ).

Figure 3 showed the life forms of the recorded species according to Raunkiaer [9]. The 69 recorded species were represented in 6 different life forms. Therophytes (30.43%) constituted the largest number of species (21 species), followed by chamaephytes (26.09%), phanerophytes (1 4.49%), hemicryptophytes (26.09%), geophytes and parasites.

Three of the recorded species were ubiquitous (have a wide ecological range of distribution).

Schouwia purpurea, Zygophyllum simplex and Zygophyllum coccineum had the highest presence values (P=73%, 68% and 60%, respectively). On

Life forms spectrum of the recorded species in the study area.

H=Hemicyptophytes, Ge=Geophytes, Ch=Chemaephytes, Th=Therophytes, Ph=Phanerophytes.

Fig. 3.

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt Floristic composition, presence value, life forms and chorology of the recorded species in Wadi Kid in South Sinai, Egypt.

P%, presence of values; Per, perennials; Ann, annuals; Ph, Phanerophytes; H, Hemicryptophyte; Ch, Chemaephytes; Th, Theophytes; G, Geophytes; P, Parasites; SA, Saharo-Arabian; SZ, Sudano-Zambezian; IT, Irano-Turanian; ES, EuroSiberian; M, Mediterranean.

Table 1.

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt Table 1.

continued

the other hand, Schouwia purpurea, Zygophyllum simplex and Reseda pruinosa showed the highest presence estimates among annuals (P = 73%, 68% and 35%, respectively). Forty-nine species (71 .01 % of the total flora) were perennials, demonstrated a certain degree of constancy. The presence of Limonium pruinosum, Salsola imbricata subsp. imbricata, Nitraria retusa and Atriplex halimus refers to salinization.

chorotypes extending their distribution all over the Saharo-Arabian, Sudano-Zambezian, Irano-Turanian, Euro-Siberian and Mediterranean regions amounted to 47.83% of the recorded flora.

However, the Saharo-Arabian chorotype (bi- and pluri-) constituted 27% and 1 6%, respectively. Thus it forms the major component of the floristic composition of this study.

Chorological affinities

Application of classification using cluster analysis to the floristic data of the coastal wadis, yielded 5 vegetation groups (Fig. 5). Ten species were recorded with variable presence values in the 5 groups. It included one tree (Acacia tortilis),

Chorological analysis of the surveyed flora (Fig. 4) revealed that 36 species (52.1 7% of the total flora) were monoregional native to SaharoArabian chorotype. Biregional and pluriregional

Classification of vegetation

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt diversity index of 1 .07±0.98 (Table 2). Stands of this group inhabited soil rich in its electric conductivity, total soluble salts, chlorides, coarse sand, fine sand, silt, and other cations such as Na, K, Ca and Mg. Sporadic species (species recorded in one stand only) were represented by 24 species or about 73% of the recorded species of this group, amongst others, Acacia tortilis, Astragalus spinosus, Blepharis edulis, Panicum turgidum, Diplotaxis harra, Teucrium polium and Zilla spinosa.

Co-dominant

Chorological analysis of the recorded species in the costal Wadi. For abbreviations, see Table 1 .

Fig. 4.

associated

species

included

Avicennia marina, Limonium pruinosum (P=27.3% for each), and Zygophyllum album (P=1 8.2%). Six species showed consistency to this group: Nitraria retusa, Avicennia marina, Limonium pruinosum, Zygophyllum album, Astragalus spinosus and Brassica tournefortii (Table 3).

Group (B):

Schouwia purpurea group This group was the most diversified among the recognized groups. It comprised of 45 species recorded from 1 0 stands, with average species richness of 1 2.7±4.1 species stands -1 , and Shannon-Wiener diversity index of 2.5±0.3. It inhabited soil with the lowest salinity (electric conductivity and total soluble salts), and lowest levels of calcium contents (Table 2). Sporadic species comprised 21 species (or about 47% of the recorded species of this group) which included, amongst others, Calligonum polygonoides, Chrozo­ phora oblongifolia, Echinops spinosus, Hyoscyamus boveanus, Iphiona scabra, Solenostemma arghel and Zizyphus spina­christi (Table 3). Co-dominant

associated species that have presence values ranged from 70-60% were Acacia tortilis, Forss­ Dendrogram showing cluster analysis of the studied 40 stands of the costal wadis, with the five vegetation groups (A-E).

Fig. 5.

shrubs (e.g.,

Fagonia arabica, Schouwia purpurea, Zilla spinosa and Zygophyllum coccineum), and annuals (e.g., Zygophyllum simplex).

Group (A):

Nitraria Zygophyllum simplex

retusa- Salvadora

persica-

group It is the largest among the separated vegetation groups. It comprised of 33 species recorded from 11 stands, with the lowest species richness of 4.7±5.4 species stands -1 , and Shannon-Wiener

kaolea tenacissima, Pulicaria undulata, Zilla spinosa, Aizoon canariense, Citrullus colocynthis, Fagonia arabica and Zygophyllum simplex.

Consistent species to this group included

Erodium oxyrhynchum, Capparis spinosa, Erodium pulverulentum, Fagonia bruguieri, Hyoscyamus boveanus, Picris cyanocarpa and Solenostemma arghel.

Group (C):

Schouwia purpurea­Zygophyllum cocci­ neum­Zygophyllum simplex group

This was the least diversified (25 species) among the recognized vegetation groups, with an average species richness of 1 0.7±3.3 species stand -1 , and Shannon-wiener diversity index of 2.3±0.3 (Table 2). The 7 stands of this group

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Mean values, standard deviations (STD) and ANOVA values of the soil variables in the vegetation groups (A-E) of the coastal wadis. EC=Electric conductivity, TSS=Total soluble salts, CS=Coarse sand, FS=Fine sand, SR=Species richness and H'=Shannon-Wiener index. **= p < 0.01 , *= p < 0.05.

Table 2.

Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt Floristic composition in the vegetation groups of coastal wadis. Figures in bold are species with highest presence values.

Table 3.

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt Table 3.

Continued.

inhabited soil with the highest clay content (higher than the total mean), high sodium and chloride contents, and lowest contents of gravel, coarse sand, fine sand and silt. Sporadic species included 1 0 species, amongst others, Bromus catharticus, Cleome amblyocarpa, Echinops hussonii, Helio­ tropium digynum and Kickxia scoparia.

Co-dominant associated 57.1 %) included Fagonia

species

(P=85.7-

mollis, Stipagrostis plumosa, Tribulus pentandrus, Iphiona scabra, Pulicaria undulata and Zilla spinosa. Two species (Echinops hussonii and Heliotropium digynum)

showed consistency to this group (Table 3).

Group (D):

Schouwia purpurea­Zygophyllum cocci­

group The 37 species in this group were recorded from 7 stands, with average species richness of 1 3.7±2.5 species stand -1 , and Shannon diversity index of 2.6±0.2. The stands of this group inhabited soil with lower content of sodium and chlorides (Table 2).

Seventeen sporadic species (ca 46% of species in this group) were recorded which included amongst others, Aerva javanica, Cleome drose­ neum

rifolia, Fagonia schimperi, Iphiona mucronata, Pergularia tomentosa and Salsola imbricata subsp. imbricata.

Co-dominant associated species that have presence values ranged between 71 .4 and 57.1 % included Calligonum polygonoides, Fagonia mollis, Stipagrostis plumosa, Citrullus colocynthis, Aspho­ delus tenuifolius and Ochradenus baccatus (Table

3). Four species showed a certain degree of consistency to this group, and included Peganum

harmala, Fagonia schimperi, Gypsophila capillaris and Salsola imbricata subsp. imbricata.

Group (E): Forsskaolea tenacissima­Iphiona scabra­ group The group size of this group was represented by 5 stands that included 43 species. The average species richness was the highest among the recognized groups with 21 .2±4.4 species stands -1 and Shannon-Wiener diversity index of 3.0±0.2 (Table 2). The stands of this group inhabited soil with the highest content of gravel, and lowest content of sodium, magnesium and chlorides.

Sporadic species included 1 3 species, of which Schouwia purpurea­Zygophyllum simplex

Cucumis prophetarum, Heliotropium arbainense, Pergularia tomentosa, Salvadora persica and Silene linearis were included.

Co-dominant associated species with presence values of 80% included amongst others Aerva javanica, Citrullus colocynthis, Pulicaria incisa, Panicum turgidum, Tribulus pentandrus and Zilla spinosa. One species ( Silene linearis) showed

consistency to this group (Table 3).

Stand ordination

Application of Principal Component Analysis (PCA) to the vegetation data (Fig. 6) revealed the segregation of the 5 vegetation groups along PCA axis 1 (Eigenvalue 0.294) and PCA axis 2 (Eigenvalue 0.074). The cumulative percentage variance of species data of the first two PCA axes were 36.8%. Stands of group (A) separated along the negative side of PCA axis 1 , while those of group (E) separated along its positive end. In the

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt The results of ordination for the four CCA axes, Inter–set correlation of the soil variables, together with eigenvalues and species – environment correlation. For soil abbreviations and units see Table 2.

Table 4.

PCA diagram showing the distribution of the 40 stands of the coastal wadis within their vegetation groups.

Fig. 6.

meantime, stands of group (B) separated along the positive end of PCA axis 2, and those of group (C) separated along its negative end.

Soil-vegetation relationships

Significant differences in the examined soil variables within the separated vegetation groups were demonstrated in Table 2. Clay, calcium, magnesium, chlorides, electric conductivity, total soluble salts, potassium and sulphates showed clear significant differences between groups at p < 0.01 and p < 0.05, respectively.

The relationship between the vegetation and soil variables was studied using Canonical Corres-

pondence Analysis (CCA). Figure 7 showed the CCA ordination biplot with vegetation groups (A-E), and the examined soil variables. It can be noted that stands of group (A) were significantly correlated with calcium, potassium and coarse sand. On the other hand, the remaining vegetation groups (B-E) cannot be easily differentiated, and tend to clump together.

The successive decrease of eigenvalues of the three CCA axes were (0.930, 0.895 and 0.224 for axes 1 , 2 and 3, respectively) that illustrated in Table 4, suggesting a well–structured data set. The species–environment correlations were higher for the first three axes, explaining 75.5% of the cumulative variance. These results suggested an association between vegetation and the measured soil parameters presented in the biplot. The inter–set correlations resulted from Canonical Correspondence Analysis (CCA) of the examined soil variables were displayed in Table 4.

CCA axis 1 was highly positively correlated with chlorides and highly negatively correlated with clay.

So this axis can be interpreted as chlorides-clay gradient. CCA axis 2 was highly positively correlated with coarse sand and highly negatively with fine sand. Thus, this axis can be interpreted as coarse sand-fine sand gradient. A test for significance with an unrestricted Monte Carlo permutation test (499 permutation) for the eigenvalue of axis 1 found to be significant (P=0.05), indicating that the observed patterns did

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Salama et al. Vegetation analysis and species diversity in the desert ecosystem of South Sinai, Egypt Correlation coefficients between soil factors and diversity indices. For soil variables abbreviations, see Table 2.

Table 5.

Canonical correspondence analysis (CCA) biplot of axes 1 and 2 showing the distribution of the 40 stands, together with their vegetation groups and soil variables.

For soil variables abbreviations, see Table 2.

Fig. 7.

not arise by chance.

The results of species diversity (species richness and Shannon's-Wiener index) were illustrated in Table 5. It was clear that, both species richness and Shannon's index showed significant differences between the recognized vegetation groups. Species richness (SR) showed significant negative correlations with EC, TSS, CS, Na, K, Ca, Mg, Cl and SO 4. Shannon-Wiener diversity index showed significant negative correlation with EC, TSS, CS, silt, Na, K, Ca, Mg, Cl and SO 4, and positively correlated with clay (Table 5).

DISCUSSION The importance of the study area from a phytogeographical point of view may be due to its position on the Sinai Peninsula, which is located in the intersection of the four phytogeographical regions: Mediterranean, Irano-Turanian, SudanoZambezian and the Saharo-Arabian region. This may reflect the relatively rich floristic diversity of the Sinai Peninsula. Chorological analysis of the floristic data revealed that the Saharo-Arabian chorotype forms the major component of the floristic structure where it was represented by more than 50%. This is in accordance with the results obtained by Danin and Plitman [30] on the phytogeographical analysis of the flora of Israel and Sinai. The presence of the monoregional Saharo-Arabian chorotype in a higher

percentage than the inter-regional chorotypes (biand pluriregionals) is not in accordance with Zohary [31 ]. The Saharo-Arabian chorotype decreased northward and replaced by Mediterranean and Irano-Turanian chorotype [30, 31 ]. This may be attributed to the fact that plants of the SaharoArabian species are good indicators for desert environmental conditions, while Mediterranean species stand for more mesic environment. The absence of the endemic species in this study is remarkable. Wickens [33] and Boulos [34] mentioned that the Saharo-Arabian region is characterized by the presence of few endemic species and genera, and absence of endemic families. Most of the endemic species in Sinai is confined to the mountain region [35].

In the Sinai Peninsula, mangrove swamps are absent from the whole stretch of the eastern coast of the Gulf of Suez (as in the western coast).

However, at the cap of the Sinai Peninsula where the Suez Gulf meets the Aqaba gulf at Ras Muhammed, there is a shallow and narrow lagoon extending from the Gulf of Suez landward. This lagoon provides a suitable site for the growth of mangal vegetation. Zaghloul [36] conducted a detailed study of Wadi Kid (South Sinai), who distinguished 3 main parts: the upstream, the main stream and the downstream; each have its specific community types that resembled the structure of the identified vegetation groups in this study. However,

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Therefore, the record of this species in Wadi Kid (along the Red Sea coast) in this study can be considered a new site for the distribution range of this species in Egypt. Most of the identified vegetation groups have very much in common with that recorded in some wadis of the Eastern Desert [37-39]. Western Desert [40-42], along the eastern [43] and western Mediterranean region [44], in South Sinai region [45-47] and in northwestern Negev, Israel [48]. The members of each pair of groups are, in some cases, linked together by having one of the dominant species in common.

Diversity is of theoretical interest because it can be related to stability, maturity, productivity, evolutionary time, predation pressure and spatial heterogeneity [49].

The present study reveals that stands of group (E) of the lowland channels of Wadi Kid (coastal wadis) that have the lowest salinity levels have the highest species richness (21 .2 ± 4.4 species stands -1 ). This may explains the high contribution of annuals in this group. The highly salinized soil with deep fine sediments (group A) dominated

by Nitraria retusa, Salvadora persica and Zygo­ phyllum simplex has the lowest species richness (4.7 ± 5.4 species stands -1 ). These results are in line with those of Abd El-Ghani and Amer [50] in El-Qaa plain of South Sinai. In this study, both species diversity measures showed high significant positive correlation (0.91 3) to each other. Species richness (SR) showed significant negative correlations with EC, TSS, CS, Na, K, Ca, Mg, Cl and SO 4. Shannon-Wiener diversity index showed significant negative correlation with EC, TSS, CS, silt, Na, K, Ca, Mg, Cl and SO 4, and positively correlated with clay.

Comparing the floristic compositions in 2 coastal wadis (Wadi Kid and El-Qaa plain) yielded 55 species in common, and 52.6% floristic similarity according to Sørensen Coefficient. This relatively high similarity may be related to the comparable soil characters of both landforms.

In Sinai, several studies have provided qualitative assessments of the distribution of plant species and associations in relation to physiographic factors in different areas of the peninsula (amongst others [4, 51 , 52]). In this study, soilvegetation relationships in the coastal wadis revealed that electric conductivity, total soluble salts, coarse sand, sodium, potassium calcium,

magnesium and chlorides were the most important soil factors along the first 2 axes of Canonical Correspondence Analysis (CCA). Whereas, gravel, coarse sand, fine sand, silt, clay, sodium and chlorides were the controlling soil variables along the first 2 axes of Canonical Correspondence Analysis. This is in accordance with the results in the present study and other relevant works such as those of El Hadidi [39], El Ghareeb and Shabana [45], Abd El-Ghani, and Amer [50] and Yair et al. [53]. According to Helmy et al. [54] who studied the distribution behavior of seven common shrubs and trees growing in southern Sinai (viz.

Retama raetam, Acacia tortilis, Moringa peregrina, Nitraria retusa, Crategus sinaica, Salvadora per­ sica, and Lycium shawii) in relation to physical

environmental factors, they also concluded that altitude, nature of soil surface, soil texture and salinity are the most important factors controlling the distribution of woody plant communities in southern Sinai. In the same direction, Mashaly [55] pointed out that moisture content, sand fraction, sodium cations, electric conductivity, and chloride contents were the most important soil factors controlling the distribution of halophytic species in South Sinai. While the contents of calcium carbonate, magnesium and calcium cations, total nitrogen, silt and clay fractions and pH were the most effective soil factors affecting the distribution of xerophytic species. These findings were also in agreement with the results obtained from this study.

ACKNOWLEDGEMENTS The authors are indebted to Prof. Dr. Taha Ramadan of the Botany Department, Assiut University for consultations in illustrations of the data Fig.s.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

In­vivo study of effects

of dithiocarbamates fungicide (Mancozeb) and its metabolite ethylenethiourea (ETU) on fresh water fish Clarius batrachus Pallavi Srivastava, Ajay Singh ,

Department of Zoology D.D.U. Gorakhpur University, India

ABSTRACT

Mancozeb belong the group of carbamate pesticides in various pest control programs. Ecotoxicological effects of Mancozeb in fishes and other non-targeted organisms not well understood. The present study investigated the effects of Mancozeb and its metabolites ethylenethiourea (ETU) on biochemicals and enzymatic parameters in Clarius batrachus. LC values (LC 50) estimate on different life stages of fish that was dose as well as time dependent. The bioassay studied after 24h and 72h during exposure with 80% of 24h of LC 50 (22.87mg/l). Protein, amino acids, glycogen, nucleic acids and enzyme succinic dehydrogenase decreased in liver and muscles, but lactic dehydrogenase levels, protease, GOT and GPT increased in the both tissues. Mancozeb is widely used fungicides in fields by the farmers and their surfaces run off goes to the water bodies and affect the life aquatic fauna. On the behalf of present study, it suggested that appropriate ecotoxicological risk assessment should make in the areas where Mancozeb is to be use in pest control activities.

Key words: Mancozeb; Clarius batrachus; ecotoxicology; LC50.

J Biol Earth Sci 201 3; 3(2): B228-B235

Corresponding author:

Ajay Singh Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur-273009, (U.P.), India Phone No. +91 8004091 424, 91 -551 22021 27 E-mail: 5september1 984@gmail.com Original Submission: 24 August 201 3; Revised Submission: 28 September 201 3; Accepted: 01 October 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B228-B235

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INTRODUCTION Mancozeb [[1 ,2-ethanediyl bis-[carbamodithioate]] (2-)] manganese is a fungicide, subclass of carbamate pesticides called dithiocarbamates. They have a similar action to carbamate insecticides they affect the nervous system through their main metabolite, carbon disulfide and ethylenethiourea.

Mancozeb marketed by the trade names Dithane, Manzeb, Nemispot, and Manzane. It uses to protect many fruit, nut, and field crops from a wide spectrum of fungal diseases. Its primary metabolite, ethylenethiourea (ETU), shown to cause thyroid and carcinogenic effects in organisms. Mancozeb, if applied to soil, will have a low mobility based on its high adsorption coefficient. If it released into water, it will tend to adsorb to sediment and suspended solids. It has low soil persistence with a reported half-life of 1 -7 days. Again, the primary concern with mancozeb is its spontaneous degradation to ethylenethiourea (ETU) in the presence of water and oxygen. ETU has a persistence of 5-1 0 weeks.

While mancozeb is practically insoluble in water making it unlikely to contaminate groundwater, its metabolite, ETU, has the potential to be mobile in soils [1 ]. The dithiocarbamates mainly used in agriculture and form part of the large group of synthetic organic pesticides that have been developed and produced on a large scale in the last 40-50 years. The worldwide consumption of dithiocarbamates is between 25000 and 35000 metric tonnes per year. Dithiocarbamates used as fungicides, being effective against a broad spectrum of fungi and plant diseases caused by fungi. In industry, they used as slimicides in water-cooling systems, in sugar, pulp, and paper manufacturing, and as vulcanization accelerators and antioxidants in rubber. Because of their chelating properties, they used as scavengers in wastewater treatment.

The general formula of dithiocarbamates characterized by the presence of:

Depending on the types of monoamines used in the synthesis of these compounds, mono or dialkyldithiocarbamates formed. Reactions with diamines result in the formation of two terminal dithiocarbamate groups linked by an alkylene

(ethylene) bridge. Both alkyl and ethylene dithiocarbamates form salts with metals, and both can be oxidized to the corresponding disulfides. Dithiocarbamates may decompose under certain circumstances into a number of compounds, such as sulfur, 5,6-dihydro-3 H-imidazol [2,1 -C]-1 ,2,4dithiazole-3-thione, ethylenethiourea (ETU), and ethylenediamine (EDA). ETU stable, has high water solubility, and is of particular importance because of its specific toxicity. For this reason, toxicological information on this compound is included in this study.

Dithiocarbamates are the disulfur analogues of carbamates, and they characterized by the presence of:

Secondary monoamines, e.g., dimethyl or diethyl amines, react with carbon disulfide to give dialkyldithiocarbamates:

Reaction with monoalkylamines gives the corresponding monoalkyldithiocarbamates. The reaction of carbon disulfide with diamines gives two terminal dithiocarbamate groups linked by an alkylene bridge:

Both alkyl and ethylene dithiocarbamates form salts with metals and both can be oxidized to the corresponding disulfides [2].

The chemical structure of ethylenethiourea (ETU) is:

During recent years, much attention has paid to the finding that ETU may occur in plant samples following the use of dithiocarbamate fungicides. It may be present in the fungicide when apply or may result from subsequent transformation [3]. ETU is

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one of the important residues in plants and in the environment following the agricultural use of ethylene bisdithiocarbamates (EBDCs). It is also a metabolite formed when EBDCs ingested by animals and man. ETU is a stable compound with respect to hydrolytic reactions but easily oxidized to ethyleneurea (EU) which is considerably more stable than ETU and can be consider major breakdown products. In animals ETU also degrade into urea, 2-imidazoline, glycine and oxalic acid. It also degrades into natural substances, which affect protein and fat. Therefore, the present study concerned with the effects of ETU (Mancozeb) in fish Clarius batrachus.

Clarius batrachus is a species of freshwater airbreathing catfish, normally lives in slow moving and often-stagnant waters in pond, swamps, streams, rivers and also in flooded rice paddies. The species spends most of its time on, or right above, the bottom surface, with occasional trips to the surface to gulp air. Economically this fish serve as great importance because of its high food values. In India, China and other countries Mancozeb is widely used in vegetables and cereals crops especially in rice field crops. Surface run-off chemicals through rain cause adverse effects by endocrine disruptors.

Fishes respond to toxicants in a manner similar to higher vertebrates. They play different roles in the trophic web, undergoing bioaccumulation of environmental pollutants. Modifications in enzyme activity confined the risk assessment of chemicals and pesticides.

Therefore, in present study different enzymes such as Lactic dehydrogenase (LDH), Succinic dehydrogenase (SDH), Protease, GOT and GPT have been checked to find out the status of internal environment of fish. Change in level of LDH shows breakdown of red blood cell and induced anemia in fish. This resulted in structural changes in liver, which is leading to the alteration of liver metabolism, and its biochemical content. SDH activity indicated anoxic hypoxic conditions when the fish exposed to toxicant and also associated with the inhibition of mitochondrial respiratory mechanisms or derangement in ultra structure and permeability of mitochondria. Protease is proteolysis enzyme, help in protein catabolism by hydrolysis of the peptide bond that link amino acids together in the polypeptide chain forming the protein. Change in GOT and GPT also confine liver damages.

MATERIALS AND METHODS Chemical

Mancozeb purchased from Syngenta India Ltd. India, and all the other chemicals were of analytical grade and obtained from Indian commercial source.

Experimented animal

The fresh-water fish Clarius batrachus (total size 1 2-1 7 cm and weight 35-50 g) for adult and for fingerlings (total size 6-8 cm and weight 9-1 2 g) brought from local fresh-water pond. They stored in laboratory tank containing 1 00 liters of de-chlorinated tap water and acclimatized to the laboratory conditions for 2 weeks.

Toxicity test

Toxicity test has performed by the method of Singh and Agarwal [4]. Five fishes kept in glass aquaria containing 1 0 L de-chlorinated tap water.

Fishes exposed for 24h to 96h to four different concentrations of pesticides in laboratory. Control fishes kept in similar conditions without any treatment. Each group of fish replicated three times.

Mortality recorded after every 24h. Dead animals removed to prevent the decomposition of body in experimental aquarium. The effective doses (LC values, upper and lower confidence limits, slope value, and heterogeneity) calculated by probit log method of Robertson et al. [5]. Product moment co-relation co-efficient was applied in between exposure time and lethal concentration [6].

Experimental designs

Fishes exposed to 80% of 24h LC 50 doses (22.87 mg/l). Experiment conducted from 24h to 72h. After completion of treatment, the test fishes removed and washed with water and killed by severe blow on head and operated their liver and muscles quickly dissected out in ice tray and used for biochemical and enzymatic analyses. Control fishes kept in similar condition without any treatment. Each experiment replicated at least 6 times and values expressed as mean ÂąSE of six replicates. Following parameters tested by different methods.

Protein level estimated according to the method of Lowery et al. [7] using bovine serum albumin as standard. Estimation of total free amino acids made according to the method of Spices [8]. Estimation of DNA and RNA performed by method of

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Piscicidal activity of fungicide mancozeb against different stages of fresh water fish Clarius batrachus at different time intervals.

Table 1.

Batches of fifteen fishes were exposed to four different concentration of the fungicides. Concentrations given are the final concentration (v/v) in the aquarium water containing de-chlorinated tap water. Values given in parenthesis are Lower and Upper confidence of LC values.

RESULTS Toxicity results

Total protein (µg/mg), total free amino acids (µg/mg), glycogen (mg/g) and nucleic acids (µg/mg) level in different tissues of fresh water fish Clarius batrachus exposure to 80% of LC 50 of mancozeb at different time intervals.

Fig. 1.

Schneider [9] using diphenylamine and orcinol reagents respectively. Glycogen estimated by anthrone method of Van Der Vies [1 0], lactic dehydrogenase by method of Anon [11 ], succinic dehydrogenase by method of Arrigoni and Singer [1 2], protease by method of Moore and Stein [1 3], GOT and GPT by method of Reitman and Frankel [1 4].

Statistical method

Two ways ANOVA performed between control and tested group. The significant was level at 0.05.

After treatment all the experimented fishes immediately settled down at the bottom of aquarium. Within 5-1 0 min, the breathing of fishes affected and they came to the water air interface for air breathing, the respiratory impairment, probably due to the effect of the pesticides on gills and general metabolisms. After 30-60 min, their swimming activity is also slow down.

Loss of equilibrium, hypo- and hyperactive activity and vertical position observed after 48h.

Finally, their activity ceases and fishes died. LC values of mancozeb for period ranging from 24-96h on both fingerlings and adult fish Clarius batrachus presented in (Table 1 ). The toxicity in both the cases was time as well as dose dependent. There was a significant negative correlation between LC values and exposure periods. Thus with an increase in exposure period the values of LC 50 of mancozeb decreased.

Biochemical and enzymatic assay

Biochemical changes in liver and muscles tissue present in (Table 2, Fig. 1 ). Enzymatic changes in liver and muscles tissue present in (Table 2, Fig. 2).

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Total protein (µg/mg), total free amino acids (µg/mg), glycogen (mg/g), nucleic acids (µg/mg) level and activity of enzyme protease (tyrosine/mg protein/h), LDH (pyruvate reduced/min/mg protein), SDH (µmoles dye/min/mg protein), GOT (µmoles pyruvate/ mg protein/h) and GPT (µmoles pyruvate/ mg protein/h) in different tissues of fresh water fish Clarius batrachus exposure to 80% of LC 50 of mancozeb at different time intervals.

Table 2.

Values given in parenthesis were percent level in parameters. * Significant (p<0.05) Two ways ANOVA was applied between control and tested groups.

DISCUSSION

Activity of enzyme protease (tyrosine/mg protein/h), LDH (pyruvate reduced/min/mg protein), SDH (µmoles dye/min/mg protein), GOT (µmoles pyruvate/mg protein/h) and GPT (µmoles pyruvate/mg protein/h) in different tissues of fresh water fish Clarius batrachus exposure to 80% of LC 50 of mancozeb at different time intervals.

Fig. 2.

Fish are ideal indicators for behavioral assays of various stressors and toxic chemicals exposure due to their constant, direct contact with the aquatic environment. Behavior provides a unique perspective linking the physiology and ecology of an organism and its environment [1 5]. Behavioral action is a sequence of quantifiable actions that operated through the central and peripheral nervous systems [1 6] and the cumulative manifestation of genetic, biochemical and physiologic processes essential to life such as feeding, reproduction and predator avoidance. For the best meet of the challenge of surviving in a changing environment, behavior allows an organism to adjust to external and internal stimuli in order to adapted environmental variables. Selective evolutionary processes

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have conserved stable behavioral patterns in concert with morphologic and physiologic adaptations [1 5]. Since behavior is not a random process, but instead of it, is a highly structured and predictable sequence of activities designed to ensure maximal fitness and survival of the individual. Fish are able to uptake and retain different toxicants dissolved in water via active or passive processes. Sub-lethal concentrations of pesticides in aquatic environments cause structural and functional changes in aquatic organisms and this is more common than mortality [1 7]. Behavioural modification is one of the most sensitive indicators of environmental stress and many affect survival [1 8]. Alterations in fish behavior, particularly in non-migratory species, can also provide important indices for ecosystem assessment. In the present study, mancozeb shows significant behavioral changes in fish (hyperactive movement, hypo movement, vertical position and loss of equilibrium). Toxicity data clearly indicate that the fingerlings are more susceptible than adult fish due to the dependence of age and body size.

It has been demonstrated that ETU degradation leads to traces of EU and other metabolites in the urine and that 1 4C-carbon dioxide. It suggested that the metabolites of ETU in the fish produced primarily by fragmentation of the imidazoline ring and decarboxylation of the fourth and fifth carbon atoms. These fragments may incorporate with polypeptide chains and alter its fate. Reduction in level of protein in experimental fish under pesticide influence is indicates hepatic insufficiency and probably malnutrition. Protein reduction might observe in the present study due to high-energy demand in TCA cycle. The decrease level was also associated with the increase level of protease enzyme in tissues. Decrease in protein content under toxicity stress has already being reported [1 9]. The decrease in total protein level and increase in free amino acids level in both tissue and liver suggest the high protein hydrolytic activity due to elevation of protease activity [20]. Increase in free amino acids level was the result of breakdown of protein for energy requirements and impaired incorporation of amino acids in protein synthesis and decline in nucleic acids level [21 ]. In the present study increase, level of amino acids and protease has observed.

The metabolites of Mancozeb in ETU, EU and natural products cause depletion of glycogen and

fat in fish body. Liver, suggested as an organ for detoxification but under the influence of toxic chemicals, the alternation of their functions may cause. During exposure to sub, lethal concentration of mancozeb fishes came under stress condition and need more energy to cope the toxicants.

Glycogen serves as reserve material. It utilize when body came under stress condition. Depletion of glycogen in liver and tissue may be due to increasement in gycolysis pathway. During stress conditions, the glycogen reserves depleted to meet energy demand [22]. The freshwater fish, Clarius batrachus, has reported to exhibit significant reduction in the level of glycogen [23] studied effects of cypermethrin on various biochemical parameters. Similar results have found in this study.

The protein is the alternative source of energy. Due to the disturbance of arrangement of tissues nucleic acid metabolism also degraded in cells, resulting in the reduction in the DNA content. Furthermore, inhibition of DNA synthesis, thus, might affect both protein as well as amino acid levels by decreasing the level of RNA in protein synthesis machinery.

The regulatory roles of nucleic acid metabolism as observed in the different animals when treated with the different pesticides. Decrease level of RNA also observed by Das and Mukherjee [24].

Metabolite of Mancozeb into glycine, affect several enzymatic pathways. In this study the level of LDH, GOT and GPT significantly increases under the effect of mancozeb. Mancozeb has ability to modify the effect of several enzymes. These enzymes are blood soluble enzyme and best indicator of stress conditions [25]. LDH may indicate changes and hypofunction of liver under the toxicants effects on the hepatocytes are in the form of tissue damage in which cellular enzymes released from the cells into the blood serum.

Increase level of LDH shown by Das et al. [24]. In the present study, the activity of SDH reduces. It is due to the mitochondrial disruption. SDH activity indicated anoxic hypoxic conditions when the fish exposed to toxicant and it was possibly, leading to decrease in the activities of oxidative enzymes and an increase in the glycolic enzymes. Narra et al.

[26] reported decreased SDH activity in different tissues of food fish Clarius batrachus exposed to chlorpyrifos. In the present study the level of GOT and GPT inhanced. It might be that GOT and GPT function at the junction between carbohydrate and protein metabolisms. Increase concentration

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showed probably elevation in gluconeogenesis through transamination of glucogenic amino acids for energy demand in stress condition. However, some pesticides caused increased transaminase activity (GPT and GOT) levels in liver and muscle tissues. Begum [27] found the activity levels of GPT and GOT increased in liver and muscle tissues of Clarias batrachus during exposed to carbofuran.

Murugesan et al. [28] also found that Sarotherodon mossambicus, when exposed to sublethal and lethal concentrations of carbaryl, showed adaptive elevation in the activity levels of GOT and GPT enzymes, particularly in liver and muscle.

CONCLUSION Our studies showed that the fungicide (Mancozeb) which is widely used in crop fields have potential to damage aquatic fauna. It is highly toxic for fingerlings and adult fish. It caused severe biochemical and enzymatic alterations in fish.

Therefore, we should avoid the extensive running off that fungicide in near water bodies.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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2. Engst R. Schnaak W. Residues of dithiocarbamate fungicides and their metabolites on plant foods.

Residue Rev. 1 974; 52: 45-67.

3. Bontoyan WR, Looker JB, Kaiser TE, Giang P, Olive BM. Survey of ethylenethiourea in commercial formulations. J Assoc Off Analyt Chem. 1 972; 55: 923-925.

4. Singh A, Agarwal RA. Possibility of using latex of euphorbiales for snail control. Science Tot Environ.

1 988; 77: 231 -368.

5. Russel RM, Robertson JL, Savin NE. POLO A new computer programme for probit analysis. Bull Entomol Soc. 1 977; AM: 20.

6. Sokal RR, Rohlf FJ. 1 973. Introduction of biostatistics.

WH Freeman and Comp San Francisco.

7. Lowry OM, Rosebrough NJ, Ferr AC, Randall RF. Protein estimation with Folin Phenol reagent. J Biol Chem. 1 951 ; 1 93: 265-275.

8. Spice JR. 1 957. Calorimetric products for amino acids. Methods in enzymology. Acad Press, pp. 468.

9. Schneider WC. 1 957. Determination of nucleic acids in tissue by pentose analysis. Acad Press New York, pp. 680.

1 0. Van der Vies J. Two methods for determination of glycogen in liver. Biochem J. 1 954; 57: 41 0-446.

11 . Anon. 1 984. Sigma diagnostics TM Lactic dehydrogenase (quantitative, colorimetric determination in serum, urine and cerebrospinal fluid) at 400-450 nm.

Procedure No. 500 Sigma chemical compan St. Louis USA. 1 2. Arrigon O, Singer T. Limitations of the phenazine methosulphate assay for succinic and related dehydrogenase. Nature. 1 962; 1 93: 1 256-1 258.

1 3. Moore S, Stein WH. A modified Ninhydrin reagent for the photometric determination of aminoacids and related compounds. J Biol Chem. 1 954; 221 : 907.

1 4. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic-pyruvic transaminases. Am J Clin Pathol.

1 957; 28: 56.

1 5. Little E, Brewer SK. 2001 . Neurobehavioural toxicity in fish. Target organ toxicity in marine and freshwater teleosts new perspectives. Toxicology and the Environment. Taylor and Francis, London and New York. pp. 1 39-1 74.

1 6. Keenleyside MHA. 1 979. Diversity and adaptation in fish behavior. Zoological physiology, Vol. 11 .

Springer-Verlag, Berlin. pp. 208.

1 7. Sancho E, Fernandez-Vega C, Ferrando MD, Andreu-Moliner E. Eel ATPase activity as biomarker of thiobencarb exposure. Ecotoxicol Environ Saf. 2003; 56: 434-441 .

1 8. Byrne PA, O'Halloran J. The role of bivalve molluscs as tools in estuarine sediment toxicity testing: A review. Hydrobiol. 2001 ; 465: 209-21 7.

1 9. Khare A, Singh S. Impact of Malathion on protein content in the freshwater fish Clarias batrachus. J Ecotoxicol Environ Monit. 2002; 1 2: 1 29-1 32.

20. Muley DV, Karanjkar DM, Maske SV. Impact of industrial effluents on the biochemical composition of freshwater fish Labeo rohita. J Environ Biol. 2007; 28(2): 245-249.

21 . Bhavan PS, Geraldine P. Biochemical stess responses in tissues of the prawn Macrobrachium malcolmsonii on exposure to endosulphan. Pest Bioch Physiol. 2001 ; 70: 27-41 .

22. Rawat DK, Bais VS, Agrawal NC. A correlative study on liver glycogen and endosulfan toxicity in Heterpneustes fossilis. J Environ Biol. 2002; 23: 205207.

23. Saha S, Kaviraj A. Effects of cypermethrin on some biochemical parameters and its amelioration through dietary supplementation of ascorbic acid in freshwater cat fish Heteropneustes fossilis. Chem.

2009; 74: 1 254-1 259.

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24 Das BK, Mukherjee SC. Toxicity of Cypermethrin in Labeo rohita fingerlings: biochemical, enzymatical and haematological consequences. Compar Biochem Physiol. (Part C). 2003; 1 34: 1 09-1 21 .

25. Palanivelu V, Vijayavel K, Ezhilarasibalasubramanian S, Balasubramanian MP. Influence of insecticidal derivative (Cartap Hydrochloride) from the marine polychaete on certain enzyme systems of the freshwater fish Oreochromis mossambicus. J Environ Biol. 2005; 26: 1 91 -1 96.

26. Narra MR, Reddy R, Kodimyala R. Effects of chlorpyrifos on enzymes as biomarkers of toxicity in Fresh water field crab Barytelphusa guerini. Int J Environ Sci 201 2; 2(4): 201 5-2023.

27. Begum G. Carbofuran insecticide induced biochemical alterations in liver and muscle tissues of the fish Clarias batrachus (Linn) and recovery response. Aqua Toxicol. 2004; 66(1 ): 83-92.

28. Murugesan R, Palaniswamy TN, Panneer S. Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) enzyme activities in different tissues of Sarotherodon mossambicus (Peters) exposed to a carbamate pesticides, carbaryl. Pesticide Sci. 1 999; 55:1 21 71 221 .

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Journal of Biology and Earth Sciences

TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

ORIGINAL ARTICLE

Seasonal variation of heavy metals accumulation in muscles of the African Catfish Clarias gariepinus and in River Nile water and sediments at Assiut Governorate, Egypt Ahmed Th. A. Ibrahim 1 , Hossam M. Omar2 1Science

and Math Department, Faculty of Education (NewValley branch), Assiut University, Egypt.

2Zoology Department, Faculty of Science, Assiut University, Egypt

ABSTRACT

Water pollution is one of the most principal environmental and public health problems in river Nile.

Heavy metals concentrations; iron (Fe), copper (Cu), cadmium (Cd), lead (Pb), zinc (Zn), chromium (Cr), manganese (Mn), mercury (Hg) and nickel (Ni) were determined in water, sediment, and fish muscles of Clarias gariepinus collected from six areas at Assiut Governorate on river Nile using inductively coupled plasma mass spectrometry (ICP-MS). The results revealed that Zn, Cu and Fe concentrations were the highest in water and muscles, followed by Mn, Cr, Pb, Cd, Ni and Hg in areas under investigation. Also, summer was the highest accumulation season and winter was the lowest one. In sediments, the order of accumulation was Fe>Zn>Mn>Cu>Ni>Pb>Cd>Cr>Hg. In general, the bioaccumulation factor (BAF) for Cr was the lowest while, the highest was recorded for Ni and Hg followed by Cu in both relation fish/sediment and fish/water. In conclusion, the present study found that the accumulation and bioaccumulation factor of heavy metals especially Zn, Fe and Cu were higher in summer season due to increase of temperature, accordingly the authors advice to decrease fish consuming in summer to avoid the accumulation of heavy metals.

Key words: Water pollutants; Sediments; muscles; factor; Assiut; Egypt.

Clarias gariepinus;

Heavy metals; Bioaccumulation

J Biol Earth Sci 201 3; 3(2): B236-B248

Corresponding author:

Hossam M. Omar Zoology Department, Faculty of Science, Assiut University, Assiut, 71 51 6, Egypt E-mail: hossameldin.mo@gmail.com Original Submission: 23 July 201 3; Revised Submission: 1 0 October 201 3; Accepted: 11 October 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B236-B248

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments

INTRODUCTION River Nile is the principal freshwater resource for Egypt, meeting nearly all demands for drinking water, irrigation, and industry [1 ]. Water pollution is one of the environmental and public health problems in Egypt [2]. Contamination of the river Nile and its tributaries with heavy metals may have devastating effects on the ecological balance of the aquatic environment and the diversity of aquatic organisms. Many articles were carried out to assess the impacts of water pollution of freshwater ecosystem on different fish species of different localities [1 , 3-5]. It will known that the level of heavy metals in the water and sediment of some parts of the river Nile is higher than the limits set by the Egyptian General Authority for Standards and Quality Control [2].

Heavy metal residues in fish flesh and its hazard effects on the health of people are a matter of great concern to food hygienists. The most toxic heavy metals of particular concern to aquatic animals are cadmium (Cd), lead (Pb) and mercury (Hg) that have the way to fish flesh mainly via gills [6, 7].

Thus, periodical evaluation of heavy metals residual level in the fish flesh and water from expected polluted area are of major importance [3, 4, 8]. The problem of metal residues in the fish flesh is serious, as reflect by the high metal concentrations recorded in the water and sediments [9]. The heavy metal residues in the tissues of Clarias gariepinus exhibited different patterns of accumulation and distribution among the selected tissues and localities. Liver was the site of maximum accumulation for the elements while muscle was the

Fig. 1.

over all areas of least metal accumulation [1 , 5, 1 0].

The African catfish, Clarias gariepinus (Burchell, 1 822), was selected as the test organism in this study for its great aquaculture and commercial value in Egypt and elsewhere in the developing world. C. gariepinus is a benthopelagic (bottom feeder), omnivorous feeder that occasionally feeds at the surface. Their diets include insects, crabs, plankton, snails and fish but also have been seen to consume young birds, rotten flesh, plants and fruits [11 ]. C. gariepinus also referred to as mudfish, is very hardy and tasty. They are able to tolerate adverse aquatic conditions where other cultivable fish species cannot survive [1 2]. It is widely cultivated and used as experimental fish [1 3].

The objective of this study was to evaluate the seasonal variation of the heavy metals residues; Fe, Cu, Cd, Pb, Zn, Cr, Mn, Hg and Ni contents of Clarias gariepinus muscles, water and sediments from six sites in Assiut governorate, Egypt.

MATERIALS AND METHODS Study sites The current work was done in six sampling areas in Assiut governorate around river Nile (Fig. 1 ). The locations were: El-Baddary City (26°58 N, 31 °23' E); Abu Tig City (27°2' N, 31 °1 9' E); Assiut City (27°1 2' N, 31 °11 ' E); Mankabad City (27°1 2' N, 31 °7' E); Manfalout City (27°1 9' N, 31 °0' E) and ElKossia City (27°26' N, 31 °51 ' E), distributed over about 84 km stretch of Assiut governorate around the Nile river with the following positions on Google earth map (Fig. 1 ).

Locations of the six selected areas under investigation.

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Samples collection

Statistical analysis

Ninety six sample from each catfish Clarias water and sediment were collected at quarterly intervals during a period of summer, 2011 to autumn, 201 2 from 6 different areas (El-Baddary, Abu Tig, Assiut, Mankabad, Manfalout and El-Kossia) (Fig. 1 ) and average depth 1 .5 m (4 samples from each site) at river Nilein Assiut governorate, Egypt. The average weight of fish samples were 260±1 2 g, while water sample was 250 ml and 1 -2 g of sediments. All samples that collected were storage in appropriate conditions until analysis.

Data were subjected to mean ± STD Err, t-test and a one-way analysis of variance using SPSS program version 1 0.0 [1 5] to perform the analysis.

gariepinus,

Sample preparation Fish flesh was minced and well mixed, then approximately 1 .0 g was placed in a 1 50 ml beaker and 1 0 ml concentrated nitric acid was added. The mixture was then heated until the appearance of dense white fumes that indicate the nitric acid had evaporated. The mixture was cooled, then 1 0 ml of 25% hydrochloric acid was added and the solution was transferred to a 250 ml volumetric flask that was subsequently brought to volume with deionized water.

Each sample of water was put in screw-capped tube till complete dryness. Ten ml of concentrated nitric acid was added to the sample to dryness then diluted to 20 ml with deionized water. The solution was filtrated, transferred to 250 ml volumetric flask, marked and stored refrigerated till analysis.

For the digestion of sediment samples, 1 -2 g wet weight is digested with repeated additions of nitric acid and hydrogen peroxide, the resultant digestate is reduced in volume while heating and then diluted to a final volume of 250 ml.

All metals concentrations in the samples were determined by ICP-MS (Thermo Fisher Scientific, Bremen, GmbH).

Bioaccumulation factor (BAF) The bioaccumulation factor (BAF) is the ratio between the accumulated concentration of a given pollutant in any organ and its dissolved concentration in water according to Authman and Abbas [1 4] using the following equation: BAF = pollutant concentration in fish tissue (mg/kg)/ pollutant in water (mg/l) or sediment

RESULTS The results of heavy metals concentration in water samples in the selected areas are given in Fig. 2. The value of Fe was ranged between (0.1 3 to 0.72 ppm); it was higher in summer of Mankabad and lower in winter of Assuit and Manfalout. The highest recorded values were less than permissible limit except in summer. The Cu values ranged between (0.09 to 0.41 ppm); it recorded the highest level in summer in El-Kossia and the lowest level in winter of Abu Tig. But, the highest value of Cu was less than the permissible limit in all areas under investigation. The concentration of Cd showed a wide range of variation between 0.01 and 0.041 ppm, which is still lower than the permissible limit in the water of all areas under investigation.

The highest value was recorded in summer of Mankabad. The concentration of Pb was lower than the permissible limit in the water of all areas under investigation and exhibited a wide range of variation between 0.009 and 0.05 ppm. The highest value was recorded in summer of Abu Tig. Zinc concentration was the highest in summer of all areas and ranged between 0.096 and 0.46 ppm. The maximum values were recorded in summer of El-Kossia. Chromium concentration was lower than the permissible limit in all areas under investigation it ranged from 0.009 to 0.05 ppm, the highest concentration was recorded in summer of Mankabad and the lowest in winter of Mankabad. All Mn concentrations values were under the permissible limit, which showed the highest value (0.06±0.002 ppm) in summer of El-Kossia and the lowest value (0.01 2±0.002 ppm) in winter of Mankabad. Mercury showed very low concentrations in all areas under investigation (0.00007 to 0.002 ppm) and its trend to accumulate was highest in summer, especially in Mankabad, but the highest value of Hg was still less than the permissible limit. Nickel values ranged between (0.0006 to 0.0073 ppm), that are lower than the permissible limit in all areas under investigation, however, the highest concentration was recorded in summer of Mankabad and the lowest in winter of Abu Tig. Table 1 showed that Zn,

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments

The seasonal variation of heavy metals accumulation in water of different areas of river Nile in Assiut governorate; A: El-Baddary, B: Abu Tig, C: Assiut, D: Mankabad, E: Manfalout and F: El-Kossia.

Fig. 2.

Cu and Fe were the highest accumulated metals in water; however, Hg and Cr were the lowest accumulated metals.

Results of heavy metal analysis of sediments from the selected areas are presented in Fig. 3. Iron represented the first highest metals in the sediment of all metals measured in the present study; however, Hg was the lowest one of accumulation.

The highest concentration of Fe was detected in autumn of El-Baddary (1 391 Âą97 ppm). Also, Cu exhibited a wide range of variation ranging from 82 to 1 97 ppm. The lowest concentration was recorded in autumn of Mankabad and the highest one was recorded in spring of El-Baddary. The concentration of Cd and Pb showed a wide range (9.0 to 32.1 and 21 .3 to 58.2 ppm respectively). The highest concentrations were detected in spring of El-Baddary. The concentrations of Mn and Zn exhibited a wide range (68.9 to 1 76.5 and 209 to 382.2 ppm respectively). The highest concentrations were detected in summer and spring of Abu Tig, respectively. The highest concentrations of Cr and

Ni were detected in autumn of El-Kossia. Nickel and Hg nearly had the same concentration and the same distribution with range from 2.02 to 5.67 ppm for Hg and from 3.38 to 7.0 ppm for Ni. On the other hand, Ni and Hg nearly had the same concentration and the same distribution (Fig. 3). Such concentrations ranged from 2.02 to 5.67 ppm for Hg and from 3.38 to 7.0 ppm for Ni. Such concentrations seem to be constant in all areas. The wide ranges of metal concentrations that, recorded for some heavy metals may be attributed to variations in mud percent and increase in heavy metals rich urban effluents draining into river. Iron, Zn and Cu showed the highest accumulated metals in sediments; however, Hg and Ni were the lowest accumulated metals (Table 1 ).

The results of heavy metals concentration in muscle of Clarias gariepinus in the selected areas are given in Fig. 4. The accumulation pattern of all heavy metals under investigation were summer> spring>autumn>winter. The concentration of Fe exhibited a wide range of variation between

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments

The seasonal variation of heavy metals accumulation in sediments of different areas of river Nile in Assiut governorate; A: El-Baddary, B: Abu Tig, C: Assiut, D: Mankabad, E: Manfalout and F: El-Kossia.

Fig. 3.

different seasons and between different areas. The highest concentration was recorded in summer of Mankabad (29.77±2.35ppm) and the lowest one in winter of Assiut (1 3.21 ppm). Fe accumulation in muscles was under the permissible limits in all areas under investigation. Copper (Cu) concentration exhibited a wide range of variation between different seasons and between different areas. The highest concentration was recorded in summer of El-Kossia (39.79±0.54 ppm) and the lowest one was recorded in winter of Mankabad (1 0.3±0.58 ppm).

The highest concentration of Pb was recorded in the muscles of fish collected from El-Baddary (2.54±0.204 ppm) in summer and the lowest in winter of the same area (0.432±0.1 5 ppm). Lead accumulation in muscles was under the permissible limits in all areas under investigation except summer of El-Baddary. The highest Cd accumulation was detected in summer of El-Kossia (1 .41 ± 0.248 ppm) and the lowest in winter of Mankabad (0.344±0.036 ppm).The highest level of Zn was recorded in the summer of El-Kossia (38.42±9.11 ppm) and the lowest concentration was observed in

winter of Mankabad (1 2.6±1 .5 ppm). A wide range of variation was recorded for Cr concentration between different seasons and different areas.

The highest level of Cr was detected in the summer of El-Baddary (0.9±0.032 ppm) and the lowest concentration was recorded in winter of Abu Tig (0.05±0.003 ppm). Also, a wide range of variation was recorded for Mn between different seasons and different areas. The highest concentration of Mn was recorded in the summer of Assiut (9.7±0.84 ppm) and the lowest in winter of Manfalout (4.8±2.01 ppm). The highest concentration of Hg was recorded in summer of Mankabad (0.37± 0.058 ppm) and the lowest in winter of Mankabad (0.06 ppm). Nickel accumulation in muscles showed a variation between different areas and seasons. The highest concentration was detected in summer of El-Baddary (1 .67±0.241 ppm) and the lowest one in winter of Mankabad (0.36±0.05 ppm). Table 1 showed that Fe, Zn and Cu were the highest accumulated metals in fish muscles; however, Hg and Ni were the lowest accumulated metals.

Bioaccumulation factor (BAF) between fish/ sediment (Table 2), showed that the highest ratio for

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments Heavy metals accumulation patterns in muscles of Clarias areas of river Nile.

Table 1.

gariepinus,

water and sediment samples from six

Fig. 4. The

seasonal variation of heavy metals accumulation in Clarias gariepinus muscles of different areas of river Nile in Assiut governorate; A: El-Baddary, B: Abu Tig, C: Assiut, D: Mankabad, E: Manfalout and F: El-Kossia.

all metals under investigation was in summer. But, on the other hand the BAF of fish/water showed a fluctuation relation according to the change of areas and seasons. Mercury and Ni showed the highest BAF in both relations (fish/sediment and fish/water) on the other hand Cr showed the lowest BAF.

DISCUSSION Sediments were considered an important indicator for environmental pollution; they act as permanent or temporary traps for material spread into the environment [1 6]. In the present study, the sediment

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Table 2. Mean

ratio of heavy metals Bioaccumulation factor (%) between Clarias gariepinus with water and sediment samples from the six areas of river Nile.

Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments accumulated heavy metals more than water; this observation was matched with Chindah and Braide [1 7] and Eja et al. [1 8]. Sediments have frequently been analyzed to identify sources of trace metal in the aquatic environment because of the high accumulation rates exhibited [1 ]. Sediment analysis allows contaminants that are adsorbed by particulate matter, which escape detection by water analysis, to be identified. The non-residual fraction of the sediment is considered to be mobile and therefore, is likely to become available to aquatic organisms [1 9].

Iron, Cu and Zinc showed the highest accumulated metals in water and sediments of areas under investigation. The reason for higher concentration of heavy metals in sediment because are rapidly removed from waters into the underlying sediments. So, sediment quality is a good indicator of pollution in water column, where it tends to concentrate the heavy metals and other organic pollutants [20]. Moreover, the significant deposition of Fe, Cu and Zn that are observed in water and sediments suggests that there is an additional enrichment of the element into different areas. The fact that Fe, Zn and Cu are essential elements taking part in various metabolic processes and enzymatic reactions they may be naturally be occurring in abundance in the environment [1 , 3, 4, 21 ].

Lead, Cd and Hg are taken up passively from the water and deposited in the organisms and hence the organisms contain more quantity of these metals than water [22]. The present study indicated the same manner of absorption of Pb, Cd and Hg by fish. This might increase the bioaccumulation level in fish and increase the actual dose of metal to which the local population is exposed [23, 24].

Chromium, Mn and Ni were lower than the permissible limit in all sites under investigation. The presence of Cr, Mn and Ni in water within the permissible level is due to its low solubility, which is corroborated by the findings of Pandey et al. [25].

These concentration values of Mn, Hg and Ni in sediment and water were in agreement with the findings by Osman and Kloas [1 0] who finally concluded that Cr, Mn and Ni in sediment and water is usually found to be lowest in a more oxidizing condition. However, Sudha Rani and Reddy [26]; Khaled [1 9] and Osman [5] they reported the enrichment of these elements in particulate matter due to the domestic and industrial waste inputs

resulted in low dissolved oxygen content with H 2S formation by bacterial activity.

In the present study, the highest levels of heavy metals in water, sediments and fish muscles were found during summer, while the lowest values occurred during winter. Similar results were obtained by Bahnasawy et al. [27] and Nwabueze and Oghenevwairhe [28]. These seasonal variations may be due to the fluctuation of the amount of agricultural drainage water, sewage effluents and industrial wastes discharged into the lake [29]. Ali and Abdel-Satar [30] attributed the increase of metal concentrations in the water during hot seasons (spring and summer) to their lease of heavy metals from the sediment to the overlying water under the effect of both high temperature and a fermentation process resulting from the decomposition of organic matter. Similar increase of metals levels in tissues of some invertebrate and fish species were observed during summer months that were related to the increased metabolism due to high temperature [1 9, 30].

Generally, heavy metal concentrations in the tissue of freshwater fish vary considerably among different studies [27, 28, 31 ], possibly due to differences in metal concentrations of water and sediments from which fish were sampled, ecological needs, metabolism and feeding patterns of fish, and also the season in which studies were carried out.

In the river, fish are often at the top of the food chain and have the tendency to concentrate heavy metals from water [32]. Therefore, bioaccumulation of metals in fish can be considered as an index of metal pollution in the aquatic bodies [33, 34] and a useful tool to study the biological role of metals present at higher concentrations in fish [35, 36].

The present study, found that highest accumulated metals in water and fish muscles of different areas were Fe, Cu and Zn. This is an agreement with the findings of Shakweer [37], who concluded that the concentration of trace metals in various organs of fish reflects the degree of water pollution in the aquatic environments in which such fish are living. Also, Ravera [38] reported that if an environment receives metal pollutants, the organisms living in it could take up the pollutants from the water or/and food and concentrate it in their bodies. Metal concentrations in fish organs exhibited seasonal variations in which all of the detected metals attained their highest levels during summer, while the lowest values were found during

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Ibrahim and Omar Heavy metal accumulation in muscles of Clarias gariepinus and in River Nile water and sediments winter. These seasonal variations were more or less similar to the fluctuation in the surrounding environment from the increase or decrease of drainage water discharged [27, 39].

Arguments regard the residuals level of heavy metals in the water and their relation to the residuals level in fish flesh. Kock and Hofer [8] reported that even low concentrations of heavy metal in the water may result in high concentrations in fish flesh. However, Wong et al. [9] reported that despite high metal levels in the seawater and sediments, concentrations of Pb, Cd and Hg in fish flesh did not exceed permissible levels. Knowledge of heavy metal concentrations in fish is important with respect to nature of management and human consumption of fish. In the literature, heavy metal concentrations in the tissue of freshwater fish vary considerably among different studies [5, 31 , 40] possibly due to differences in metal concentrations and chemical characteristics of water from which fish were sampled, ecological needs, metabolism and feeding patterns of fish and also the season in which studies were carried out. In the river, fish are often at the top of the food chain and have the tendency to concentrate heavy metals from water [32].

Iron, Cu and Zn are coenzymes and is necessary for the synthesis of hemoglobin [41 ] but very high intake of these elements can cause adverse health problems. The accumulation trend of Fe, Cu and Zn were approximately summer> spring>autumn>winter. The elevation of these metals accumulation in this study may be due to industrial and sewage wastes. Also, it may be due to elevated metal-binding protein synthesis as recorded by Yacoub [41 ]. In addition, the ability of copper can combine with other contaminants such as ammonia, mercury and zinc to produce an additive toxic effect on fish [41 ]. Mining smelting and sewage disposal are major source of zinc pollution. Fish take it up directly from water [42].

The accumulation of these metals in muscles was under the permissible limits in all sites under investigation, except for Cu in summer of some areas. Similarly, Tayel et al. [43] observed the increase of the three previous metals accumulation in fish muscles and returned that to the increase of total dissolved metals in Nile water and sediments.

The presence of higher concentrations of Fe, Cu and Zn in different fish species due to exposed to higher concentration levels of these elements

through water, food and sediments are not suitable for human consumption [27, 33, 44].

Cadmium, Pb and Hg are non-essential element and higher concentrations can occur in aquatic organisms close to anthropogenic sources. These metals are toxic even at low concentrations and have no known function in biochemical processes [42]. The increase of these metals level are due to the discharge of industrial, sewage and agricultural wastes in the present investigated areas. High accumulation of heavy metals in muscles may be due to its strong binding with cystine residues of metallothionein [43]. According to the present result of these metals accumulation, level did not exceed the permissible limits except for Pb in summer of Mankabad (2.54 ppm). The increases in Pb level may be attributed to high Pb concentration in water [43]. Thehigh concentration of Cd in muscles of C. gariepinus may be associated with the life style of the species spending more time at the bottom and or muds [1 9, 45-47]. The major health impacts caused by Hg level affect people who are not working directly in Hg related industry but who have a regular fish diet that has Hg residues above the permissible limit [48]. The differences in metal concentration, which was observed between different areas and the amount, depend on the concentration in the surrounding environment, feeding habit and adaptability of the species to the chemicals and mechanisms developed by the species to biotransformation [47].

Manganese, Cr and Ni functions as essential constituent for bone structure, reproduction and normal functioning of the enzymes system. They are toxic only when present in higher amount, but at low level is considered as micronutrient [42].

According to the present result of these metals accumulation, level did not exceed the permissible limits. The accumulation pattern of these metals were summer>spring>autumn>winter. Generally, the uptake and bio accumulation of heavy metals in the target organs are also influenced by the biochemical functions of the tissue or organ and physiological processes that go on in them, some of which occur in an intricately complex network of interrelationships [49]. When fishes are exposed to elevated levels of metal polluted aquatic ecosystem, they tend to take those metals up from their direct environment. It is assumed that most metals are taken up in the ionic form and influenced by various environmental factors such as pH and

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In the present study, the levels of heavy metals in different fish muscles showed highly significant differences between seasons. The level of the nine investigated metals was significantly higher in the muscle tissues in summer compared with other seasons (t-value=-3.235, -8.422 and -2.21 5; P= 0.001 , 0.003 and 0.023 for comparison of summer value with autumn, winter and spring seasons respectively). Similar increase of metals levels in tissues of fish species were observed during summer that were related to the increased metabolism [1 9, 27]. The measured metals attained their maximum values during summer, while their lowest values were found during winter. The concentration of metals in the surrounding water was also higher in summer and lower in winter.

These results were in agreement with Bahnasawy et al. [50] and Bahnasawy et al. [27]. On the other hand, Ansari et al. [51 ] reported that variations of the metals concentration at a given area may be often due to seasonal changes of the organisms' tissues weight rather than to any variability in the absolute metal content of the organism. The seasonal variations of heavy metals in fish were reported by many authors [27, 50, 52, 53].

The level of heavy metals recorded in muscles in this study was generally low when compared with the limit of chronic reference values suggested by EOS [54] and ELP [55] except for Cu and Pb in the hot seasons in some areas under investigation.

From the present result, we can suggest that increases of heavy metals concentration in muscles may be returned to its accumulation in the lipid layer under skin. This suggestion agrees with many authors who reported that muscle is not an active tissue in accumulating heavy metals [52, 56-58].

Skin is an important excretory organ for heavy metals with the mucus [59]. Thus, the reason for high metal concentration in the skin could be due to the metal complexion with the mucus that is impossible to be removed completely from the tissue before the analysis [58].

Canterford et al. [60] reported that, it is useful to express results in terms of BAF when comparing the order of uptake of metals. Bioaccumulation factor between fish/sediment (Table 2) showed that the highest ratio for all metals under investigation was in summer. But, on the other hand the BAF of fish/water showed a fluctuation relation according to the change of areas and seasons. The observed

high BAF indicates that fish has a high potential to concentrate heavy metals in their muscles [1 8, 61 , 62]. Also, Chindah and Braide [1 7] found high concentration of Fe and Cu in three species of fish and sediment from creek. In addition, Chindah et al.

[63] recorded high concentration of Cr, Cd, and Pb in the tissues of shrimps (Penaeus notialis). Other studies of Alfred- Ockiya et al. [64] as well as Ovuru and Alfred-Ockiya [65] reported the same observations. The BAF for Cr was generally observed to be the least while highest BAF was recorded with Ni and Hg followed by Cu in both relation fish/sediment and fish/water. Previous studies supported this observation and showed that metals such as Cd bioaccumulated poorly in fish [1 , 66]. The obtained results in Table 2 suggested that BAF of heavy metals in fish muscles with water in summer displayed the highest metal concentrations. Also, the BAF of heavy metals in fish muscles with sediments in hot season displayed the highest metal concentrations. This confirms the previous findings on Assiut governorate since it receives huge amounts of sewage, industrial and agricultural wastes, which collects these pollutants from different districts through its way from Assiut.

Fish surviving at highly polluted areas accumulate higher levels of heavy metals than those surviving at less polluted area of the same place [1 , 3, 4, 67, 68]. The variation that existed between the heavy metals and different season of C. garie足 pinus can be attributed to the fact that the different metals have different rate of absorption in tissues of C. gariepinus [1 ] and that metals have different sources of entry into the water body and consequently bioaccumulating in the clam [69]. In conclusion, the present study found that the accumulation and bioaccumulation factor of heavy metals especially Zn, Fe and Cu were higher in summer season due to increase of temperature, accordingly the authors advice to decrease fish consuming in summer to avoid the accumulation of heavy metals.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Cisplatin and/or etoposide induces oxidative stress in testicular, hepatic and kidney tissues in male albino mice U. Kanchana Ganga1 , B. Kishori 1 , P. Sreenivasula Reddy2 1Department

of Biotechnology, Sri Padmavati Mahila University, Tirupati, A.P., India of Zoology, Sri Venkateswara University, Tirupati, A.P., India

2Department

ABSTRACT

Cisplatin and etoposide are used to treat malignancies. The aim of the present study was to investigate the adverse effects of these anti-cancer drugs on tissue oxidative status in male mice. Healthy male mice were divided into four groups, each consisting of eight animals. The first group was served as control and injected with saline; animals in the groups 2 and 3 were injected with cisplatin and etoposide respectively. The animals in group 4 were administered with both cisplatin and etoposide. Injections were given intraperitoneally three times per week for 35 days on 1 st, 3rd and 5th day of the week.

Animals were sacrificed by cervical dislocation on 36th day, tissues were isolated and analyzed for oxidative stress. Significant decrease in tissue indices was observed in mice injected with cisplatin and/or etoposide. The activity levels of superoxide dismutase and catalase were significantly decreased with an increase in levels of lipid peroxidation in testicular, liver and kidney tissues of experimental mice when compared to controls.The results suggest that the administration of anticancer drugs such as cisplatin and/or etoposide during chemotherapy induces oxidative stress in all tissues. To mitigate the oxidative stress during chemotherapy, it is hereby advised to co-administer anti-oxidants to the patient undergoing chemotherapy.

Key words: Anticancer drugs; Organ indices; Antioxidant enzymes; Male albino mice.

J Biol Earth Sci 201 3; 3(2): B249-B254

Corresponding author:

Battini Kishori Department of Biotechnology, Sri Padmavati Mahila University, Tirupati, A.P., India E-mail: kktinku@rediffmail.com Original Submission: 30 July 201 3; Revised Submission: 1 8 October 201 3; Accepted: 25 October 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B249-B254

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Kanchana Ganga et al. Cisplatin and/or etoposide induces oxidative stress in male albino mice

INTRODUCTION Cisplatin and Etoposide are widely used chemotherapeutic agents for the treatment of various cancers, such as, testicular, ovarian, bladder, liver, lung, head and neck, uterine and cervix carcinomas and leukemia [1 -4]. Cisplatin (cis-diamminedichloroplatinum II) (CDDP) is platinum-derived, DNA alkylating agent [5]. Etoposide is a semisynthetic epipodophyllotoxin agent usually used in combination with other anticancer drugs. Etoposide is a topoisomerase II inhibitor [6] that forms a ternary complex, DNA–topo II–ET, which in turn prevents the ligation of the double-strand breaks [7, 8] resulting in chromosomal aberrations [9]. Though these are effective in controlling carcinomas, cisplatin and etoposide treatments cause severe hepato[3, 1 0-1 2], nephro- [11 , 1 3-1 7] and testicular toxicity [1 8-24]. The alterations in the testis, kidney and liver functions induced by anticancer drugs are closely associated with formation of reactive oxygen species (ROS) in the tissues [1 0, 1 2, 1 5, 1 7, 25].

When aerobic organism cells are exposed to several endogenous and exogenous agents, reactive oxygen species (ROS) such as singlet oxygen (O •), hydrogen peroxide (H 2O 2), hydroxyl radical (OH), superoxide anion (O 2-), and peroxyl radicals are produced which in turn causes damage to nucleic acids, proteins, lipids etc [26]. Lipid peroxidation levels were used as marker for oxidative damage in cells and tissues [27]. Lipid peroxidation is analyzed in biological samples by measuring the quantity of malondialdehyde. To prevent damage from ROS, cells possess different antioxidant enzymes such as glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), catalase (CAT) and glutathione S transferase. The present study was aim to investigate the effect of administration of cisplatin and etoposide alone or in combination on lipid peroxidation and selected anti-oxidant enzyme activities in liver, kidney and testis of male Swiss albino mice.

MATERIALS AND METHODS Animals

Male Swiss albino mice (28 ± 5 g) were obtained from an authorized vendor (M/S Raghavendra Enterprises, Bengaluru, India). The animals were housed (four per cage) in polypropylene cages (1 8” x 1 0” x 8”) lined with autoclaved paddy husk as

bedding material, and were provided with standard laboratory chow (obtained from HLL Animal Feed, Bengaluru, India) and tap water ad libitum. The animals were maintained at temperature 24 ± 2˚C under a well regulated dark and light (1 2:1 2 h) schedule. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA 2003), Government of India. The protocol was approved by the Institutional Ethical Committee of Sri Venkateswara University.

Chemicals

Cisplatin and Etoposide were purchased from local drug store, Tirupati. All other chemicals used for various assays were of analytical grade and obtained from local commercial sources.

Experimental design

Healthy male mice were randomly allocated into four groups each consisting of eight animals. The mice in the first group were served as control and received normal saline only. The animals in the groups 2 and 3 were injected with cisplatin and etoposide respectively. The animals in the fourth group were administered with both cisplatin and etoposide. Cisplatin and etoposide were injected intraperitoneally at doses 3 mg and 4 mg/Kg body weight respectively. Injections were given three times per week for 35 days on 1 st, 3rd and 5th day of the week.

Control group were given an equal volume of the vehicle alone. These doses were selected based on earlier literature [22]. On 36th day of experiment, animals were sacrificed by cervical dislocation. Testis, liver and kidney were isolated, cleared from adhering fluid, weighed to the nearest milligram and used for biochemical analysis. Tissue somatic index (TSI) was calculated using the following formula: TSI = [weight of the tissue (g) / body weight of the animal (g)] x 1 00

Determination of lipid peroxidation

A breakdown product of lipid peroxidation, thiobarbituric acid (TBA) reactive substance was determined by the method of thiobarbituric acid reaction [28]. A standard curve was constructed with the known amount of malondialdehyde and the absorbance was measured at 532 nm.

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Assay of SOD and catalase

Superoxide dismutase (EC 1 .1 5.1 .1 ) (SOD) was assayed in the microsomal fraction according to its ability to inhibit the autooxidation of epinephrine at alkaline medium [29]. The activity of SOD was expressed in units per min per mg protein. Catalase (EC 1 .11 .1 .6) activity was determined based on its ability to decompose H 2O 2 [30].

The activity of catalase was expressed in nmol of hydrogen peroxide decomposed per min per mg protein. Protein content in the enzyme source was estimated by the method of Folin Phenol Reagent [31 ] using bovine serum albumin as standard.

Effect of treatment of cisplatin, etoposide alone or in combination on levels of MDA (n moles of malondialdehyde formed/gram tissue) in liver (solid bar), testis (striped bar) and kidney (open bar) of mice.

Bars are mean ± SD for 8 animals per group. *indicates statistically significant at p<0.05 against control.

Fig. 1.

Effect of treatment of cisplatin, etoposide alone or in combination on activity levels of SOD (units/gram protein/minute) in liver (solid bar), testis (striped bar) and kidney (open bar) of mice.

Bars are mean ± SD for 8 animals per group. *indicates statistically significant at p<0.05 against control.

Fig. 2.

Statistical data analysis

All data were expressed as mean ± SD of eight animals per group. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-test using SPSS (student version 7.5, SPSS Inc., UK). Probability values ≤ 0.05 were taken as statistically significant.

RESULTS The relative weights of liver, kidney and testis were presented in Table 1 . No significant change in indices of liver and kidney were noted following cisplatin and etoposide administration in mice. In contrast, a significant (p<0.05) decrease (-65.30%) in the weight of testis was observed in mice injected with anticancer drugs when compared to controls (Table 1 ). MDA is an important indicator of lipid peroxidation. Administration of cisplatin and etoposide alone or in combination showed an increase (5.98-22.31 %) in testicular MDA content (Fig. 1 ) relative to control rats. Significant increase in MDA content was also observed in liver and kidney of experimental mice (Fig. 1 ). The activity of catalase and SOD showed significant decrease in the experimental mice (Figs. 2, 3) when compared to the control mice.

DISCUSSION Current research investigations are targeted to understand the possible side effects of widely used

Effect of treatment of cisplatin, etoposide alone or in combination on activity levels of catalase (nmoles of H 2O 2 metabolized/gram protein/minute) in liver (solid bar), testis (striped bar) and kidney (open bar) of mice.

Bars are mean ± SD for 8 animals per group. *indicates statistically significant at p<0.05.

Fig. 3.

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Kanchana Ganga et al. Cisplatin and/or etoposide induces oxidative stress in male albino mice Changes in the relative weights (w/w %) of liver, kidney and testis following treatment with cisplatin and/or etoposide.

Table 1.

Values are mean Âą S.D of 8 animals Values in parentheses represent % change from control Mean values in same superscript in a row are not significantly different. P < 0.05.

anti-cancer drugs in human health and disease management. Considering the variety and chemical nature of anti-cancer agents known, investigations on their role in tissue oxidative status remain limited. Commercial availability of drugs has made them as dependable sources for use in specific research investigations. A systematic study employing cisplatin and/or etoposide investigating their effects on tissue oxidative status has not been reported. Since oxidative status is known to affect several physiological processes in tissues, this study therefore focused on the effect of cisplatin and etoposide alone or combination on levels of lipid peroxidation and anti-oxidant enzyme activities in liver, kidney and testis of mice.

Administration of cisplatin and/or etoposide induced lipid peroxidation and decreased the activities of SOD and catalase in mice liver, kidney and testis. Lipid peroxidation, an indicator of oxidative stress in tissues cause peroxidative damage of cellular lipid content and is assessed through estimation of thiobarbituric acid-reactive substances (TBARS) in a tissue. Malondialdehyde (MDA) being the end product remains an important molecular marker of lipid peroxidation. MDA is also used as an indicator of oxidative damage in various diseases [32, 33]. Generation of the reactive oxygen species within a tissue is neutralized by the antioxidant enzymes present in the tissue. Increase in the tissue MDA content detected was therefore indicative of the oxidative stress induced by cisplatin and/or etoposide. This was further affirmed by the noted decrease in the antioxidant enzyme activities superoxide dismutase and catalase. Our results are in agreement with the earlier reports on induced lipid peroxidation and decreased SOD and CAT levels by treatment with anticancer drugs [1 6-1 7,

24, 34-36].

Lipid peroxidation is a key process in many pathological events and it is induced by oxidative stress. Lipid peroxidation is regarded as one of the fundamental mechanisms of cellular damage caused by free radicals having reacted with lipids causing peroxidation that eventually results in the release of products such as malondialdehyde (MDA), hydrogen peroxide (H 2O 2) and hydroxyl radicals [26, 37, 38]. The results of the present study indicated that lipid peroxidation in liver, kidney and testis significantly increased in the tissues of cisplatin and/or etoposide administered mice.

Superoxide dismutase catalyses the dismutation of superoxide anion radicals to hydrogen peroxide and in turn the catalase (CAT) degrades hydrogen peroxide into a molecule of oxygen and water [39].

Lowered activities of SOD and CAT resulted in the accumulation of these highly reactive free radicals and eventually generate reactive oxygen species that lead to detrimental effects in different tissues.

This damage occurs due to imbalance between reactive oxygen species generation and antioxidant system. The present results are in agreement with previous studies, which reported the induction of oxidative stress and lipid peroxidation in anticancer drug treated animals [11 , 1 5-1 6, 24, 35, 40-41 ].

CONCLUSION The results of the present study indicate that lipid peroxidation in liver, kidney and testis was induced in mice after cisplatin or etoposide treatment. The results further suggest that the activity levels of SOD and catalase in the tissues significantly decreased after anti-cancer drug(s) administration. These changes were more pronounced

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Kanchana Ganga et al. Cisplatin and/or etoposide induces oxidative stress in male albino mice in mice after administration of both cisplatin and etoposide. The results give caution to proceed for further research towards the reduction of side effects by combination of anticancer drugs with antioxidants.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

AUTHORS' CONTRIBUTIONS PSR and BK conceived the idea, designed and supervised the work. UKG participated in designing the study, carried out the treatment of animals, and performed enzyme assays and biochemical analysis. All authors are involved in drafting the manuscript, read and approved the final version of the manuscript.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the partial financial support received from the Department of Biotechnology, New Delhi. Technical help received from D.V.L.N. Kalpalatha (DBT in-house project fellow) is gratefully acknowledged.

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1 6. Noori S, Mahboob T. Antioxidant effect of carnosine pretreatment on cisplatin-induced renal oxidative stress in rats. Ind J Clin Biochem. 201 0; 25: 86-91 .

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1 8. Stephenson WT, Poirier SM, Rubin L, Einhorn LH. Evaluation of reproductive capacity in germ cell tumor patients following treatment with cisplatin, etoposide, and bleomycin. J Clin Oncol. 1 995; 1 3: 2278-2280.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Trizole: a new fungicidal group induced chromosomal aberrations in Asian Catfish (Clarius batrachus) Pallavi Srivastava, Ajay Singh Department of Zoology, D.D.U.University Gorakhpur, India

ABSTRACT

Chromosomal studies have received considerable attention in recent years, in part from a growing interest in the evaluation of genotoxicity of environmental toxicants and carcinogens. Chromosomal aberrations induced by Propiconazole (trizole) has been observed in kidney tissues of freshwater Asian Catfish, Clarius batrachus. A control group of fish was maintaned without any treatment, positive control group was treated with mitomycin-C and two experimental groups were exposed to sublethal concentrations of Propiconazole (1 .11 mg/l and 2.23 mg/l). After 24h, 48h, 72h, and 96h the mitotic index in kidney tissue was significantly decreased (p<0.05) in both treated groups. These findings thus indicate that trizole fungicides are able to induce genotoxic effect in catfish.

Key words: Chromosomal aberrations; Asian Catfish; Clarius batrachus; Propiconazole; Trizole.

J Biol Earth Sci 201 3; 3(2): B255-B260

Corresponding author:

Dr. Ajay Singh Department of Zoology, D.D.U.University Gorakhpur, India Phone 91 -551 22021 27 E-mail: 5september1 984@gmail.com Original Submission: 02 October 201 3; Revised Submission: 02 November 201 3; Accepted: 08 November 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B255-B260

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Srivastava and Singh Trizole: a new fungicidal group induced chromosomal aberrations in Clarius batrachus

INTRODUCTION In view of fast growing agricultural applications of chemicals, fertilizers and pesticides, the evaluation of their genotoxic potential is necessity to conduct adequate hazard and to produce safer and sustainable aquatic environment. Trizole refers to either one of a pair of isomeric chemical compounds with the molecular formula C 2H 3N 3, having 5-membered ring of two C-atoms and 3-N 2 atoms.

The two isomers are: 1 ,2,3-trizole and 1 ,2,4-trizole.

1 ,2,3-trizole

1 ,2,4-trizole

Trizoles are relatively stable functional groups and trizole linkages can be used in variety of applications (for e.g. replacing the phosphate backbone of DNA).

In agriculture trizole containing fungicides immerge as knight armor in pest management. Trizole containing fungicides was used as antifungal in agriculture for increasing food crops [1 ]. They used in broad spectrum for Ascomycetes, Basidiomycetes, and Deuteromycetes diseases on wheat, barley, rye, oats, rice, and grasses grown for seed, turf and ornamentals. Trizole, belonging to the DMI (demethylation inhiitors) group with rapid acropetal systemicity acts on the pathogen inside the plant to stop disease development by interfering with sterol biosynthesis in fungal cell membrane. The trizole fungicides inhibit one specific enzyme C1 4-demethylase, which plays a role in sterol production.

Sterol such as ergosterol, are needed for membrane structure and functions, making them essential for development of functional cell wall. Therefore, these fungicides result in abnormal fungal growth and eventually death.

Although trizole containing fungicides have a shorter half-life and lower bioaccumulation but announced effects on the aquatic ecosystems may arise from spray drift or surface run-off [2]. They have been reported to undergo transformation of secondary metabolites in terrestrial mammals [3].

Series of study shows that the trizole, Propiconazole (PCZ) altered the metabolic pathways, cell signaling, cell growth pathways, cell cycle genes

and other transcriptional factors [4-6].

Propiconazole, (1 -[[2-(2,4-dichlorophenyl)-4-propyl-1 ,3-dioxolan-2-yl]methyl]-1 H-1 ,2,4-triazole) is a trizole group fungicides which was introduced in mid 1 970s classified as category III and IV. These fungicide is highly effective against many different fungal diseases, especially powdery mildews, rust and many leaf spotting fungi. It also used in stored grain.

In body tissues it metabolite into 1 ,2,4-trizole, trizole alanine, trizole acetic acids, trizole pyruvic acid and trizole lactic acid. Of these metabolites 1 ,2,4-trizole and trizole alanine are the main metabolites which have toxic effects [7]. However, in spite of their widespread use genotoxic evaluations have been performed in this studies.

MATERIALS AND METHODS Chemical

Propiconazole (PCZ) (CAS No. 60207-90-1 ) has purchased from Syngenta Ltd. from India, a technical grade pesticide. Other chemicals such as Giemsa stain has purchased from local Indian market.

Experimented animals

The fresh-water fish Clarius batrachus obtained from local hatchery (Chhappy Hatchery). Fish had an average weight of 40.01 ±1 .50 g and average length 1 7.26±0.1 0 cm for adult and for fingerlings, the average weight had 1 0.20±1 .45 g and average length 8±0.1 0 cm. Fish were fed with commercial fish food, and acclimatized under laboratory conditions for 2 weeks, containing de-chlorinated tap water (pH = 7.6, alkalinity 1 50 mg/l CaCO 3, DO = 7.03 mg/l, Temperature = 22°C). The photoperiod used 1 2/1 2 dark/light. The aquaria used in study was made of glass had constant aeration.

Physical dimensions of aquarium had 1 00×40×40 cm, and a 1 20L capacity.

Chromosomal aberration test

Fish divided into 4 groups and each group containing 1 0 individual fish. Group 1 : Negative control (only tap water was used), Group 2: Positive control (4 mg/l Mitomycin C, Sigma), Group 3: 1 .11 mg/l PCZ, Group 4: 2.23 mg/l PCZ.

Tissue preparation

Chromosomal aberration test has done by the

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Srivastava and Singh Trizole: a new fungicidal group induced chromosomal aberrations in Clarius batrachus method of [8]. Take healthy fish (60-1 00 g). Inject 0.05% Colchicine intramuscularly 1 ml per 1 00g of body weight. Kept, fish alive for 1 .5-2 hrs after injection of colchicines. Anesthetize fish with ethylene glycol, dissect out the kidney tissue in a Petri dish, and cut into small pieces. Homogenize tissue in 6-8 ml hypotonic solution in homogenizer. Pour the cell suspension in 1 5 ml centrifuge tube and incubate for 20-25 minutes at room temperature.

Stop the hypotonic action by adding 1 ml freshly prepared Conroy’s fixative and left for 30 minutes or overnight. Mix it gently with pasture pipette. Centrifuged cell suspension at 1 200-1 500 rpm for 1 0 minutes at room temperature. Removed supernatant with a pipette and slowly over layer 6-8 ml freshly prepared chilled fixative. Keep the tube in refrigerator for half an hour. Mix contents and centrifuge cell suspension at 1 200-1 500 rpm for 1 0 minutes at room temperature. Remove supernatant without disturbing cell pellet at the bottom, add fresh fixative, and keep the tube in refrigerator for half an hour. Repeat this step 3-5 times until transparent cell suspension is obtained. Take cell suspension in pipette and dropped it onto grease free, pre cleaned slide. Allow the slide to air/flame dry. Keep the slides for ageing (1 -3 days). Stain with 4-5% Giemsa in phosphate buffer (pH 6.8) for 1 5-20 minutes.

Washed with DDW and air-dried. Make permanent preparation by mounting in DPX. Screen in oil immersion objective (1 00x).

the maximum decrement in mitotic index in kidney tissue has founded after 48h in comparison to 24h, 72h and 96h with respect to negative control. It became 26.66, 29.33, 21 .33 and 1 8.66 24h, 48h, 72h and 96h respectively in Group 4 (2.23 mg/l PCZ).

Group 4 showed more of decrement than Group 3 in all periods but followed similar trend as Group 3.

The results were similar to those of positive control group also. In Groups 3, 4 and in positive control the frequencies of Chromosomal break, chromatid breaks, and deletions founded significantly increased after 24h and 48h (Table 1 ).

DISCUSSION The results of present study showed the induction of chromosomal aberrations in kidney tissue of Clarius batrachus when exposed to different concentrations of PCZ. In line with our findings, a number of workers have reported genotoxicity studies in different species of fish using cytogenetic analysis. An advantage of chromosomal studies is that they reveal a measure of sub-lethal effects of xenobiotics in vivo. In epidemiological studies, it has been shown that people with elevated frequencies of chromosomal aberrations (CA) in their peripheral blood lymphocytes have a significantly elevated risk A

B

C

D

Statistical analysis

The Mitotic Index (MI) was calculated. Then the mean± standard error for each group was calculated. Student’s t-test employed for comparison of control and experimental animals [9].

RESULTS The result of chromosomal aberrations and mitotic index in kidney tissue of Clarius batrachus exposed to 1 .11 mg/l and 2.23 mg/l of PCZ concentrations for 24h, 48h, 72h and 96h were shown in (Table 1 , Fig. 1 ). In both of the groups receiving PCZ as well as in the positive control group also, Mitotic Index (MI) was calculated. Significant decrement (p<0.05) in MI was observed in both treated groups and positive control group when compared to negative control group. The MI was 1 6.44, 22.66, 1 8.22 and 1 5.1 0 after 24h, 48h, 72h and 96h respectively in Group 3: (1 .11 mg/l PCZ). In Group 3,

a c b Showing the Chromosomal Aberration (CA) in kidney tissues of fish Clarius batrachus. A = after 24h treatment, B = after 48h treatment, C = after 72h treatment and D = after 96h treatment. a = acentric chromosome, b = ring chromosome, c = chromatid deletion.

Fig. 1.

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Srivastava and Singh Trizole: a new fungicidal group induced chromosomal aberrations in Clarius batrachus Table 1.

Frequency of chromosomal aberrations induced by Propiconazole in kidney tissues of Clarius

batrachus.

A: Chromatid gap, B: Chromosome gap, C: Chromatid break, D: Chromosome break, E: Chromatid deletion, F: Fragment, G: Acentric fragment, H: Ring chromosome, I= dicentric chromosome * Significant (P<0.05) when Student’s ‘t’ test was applied between treated and control groups.

of developing cancer [1 0-1 4]. CA is small fraction of a huge amount of changes in chromosomal DNA and reflects an enormous plasticity of the genome, which has far-reaching consequences for evolutions [1 5]. This report has focused on the CA caused by fungicides. After exposure of fungicide the formation of Chromosomal break, chromatid breaks, ring chromosomes, fragments, acentric fragments and deletions observed. Results for exposure of PCZ for 24h to 96h showed that these CA may be effective in detecting induced genotoxicty and showed that the extensive use of these trizole-containing fungicides as dangerous. It noted that some authors concluded that the magnitude of the genotoxic response correlated with duration of exposure.

However, the exact mechanisms are still unknown.

However, it also well documented that age is significantly associated with an increase in the occurrence of MN (micronuclei) and CA (chromosomal aberration [1 6-1 8]. Eukaryotic chromosomes are uninemic. They contain one continuous, DNA molecule in the pre-synthetic phase of cell cycle, which is replicated during the S-phase. During S-phase, these DNA molecules are extremely long and fibrillar structure. While in metaphase chromosome is about 1 0µm long. These packaging of chromoso-

mes are associated with various types of protein.

Due to their enormous dimensions, DNA molecules in chromosome are permanent targets of chemical and physical damage of diverse origin such as chromosomal aberrations [1 9]. Experimental analysis has shown that CA, in fish and other organisms, induced by DNA strand breaks (DSBs) [20-22].

DSBs arise spontaneously or through a variety of cellular processes. Sources of spontaneously induced DSB are DNA replication and DNA excision repair. Error in both mechanisms induced CA. The majority of chemical mutagens are not able to induce DSB directly but leads to other lesions in chromosomal DNA, which during repair, or DNA synthesis, may give, and rise to DSB and eventually to CA. DSBs, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. DNA double strand breaks are potentially lethal to cells. These breaks have a number of causes including replication through a single-strand nick, chemical damage from reactive oxygen species or ionizing radiation, enzyme damage and mechanical stress. Mistakes made in the repair of DNA damage can lead to genomic instability and hence give rise to cancer [1 9].

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Srivastava and Singh Trizole: a new fungicidal group induced chromosomal aberrations in Clarius batrachus There are two main avenues for the repair of double breaks. The first is non-homologous end joining (NHEJ). NHEJ is used by cells to join broken ends by simply religating them. However, this process is not perfect and sometimes incorrect ends are joined to give rise to chrmosome translocations.

When the chemical moieties terminating at the break are not easily rejoined or polymerase activity can give rise to mutations i.e. CA. Various types of translocational aberrations (dicentric cromosome, ring chromosome) have observed in present study by the exposures with fungicides in kidney tisues of fish Clarius batrachus, and second is Homologous Recombination (HR). HR pathways allows a damaged chromosomes to be repaired using a sister chromatids (available in G2-phase after DNA replication) or homologous chromosomes as a tempelate. It has been reported that HR, the most accurate process, depends upon the Rad52 and Rad 51 proteins which encoded by the genes BRCA1 and BRCA2. Mutation in these genes products due to exposures of chemicals and pesticides leads [1 9] different types of CA suah as exchange types of CA like reciprocal translocations, Chromatic gap and deletion, dicentrics and corresponding fused acentric fragements, which also osresved in present studies by the exposure of Propiconazole.

CONCLUSION It is evident that multiple molecular mechanisms can lead to the induction of different types of CA. Mitotic index (MI) provides a useful index of accumulated genetic damage during the lifespan of the cells. The MI in kidney tissue of fish evaluates the kinetics of cytogenetic alterations under fungicides influence. So based on the present study we can say that there have a positive association between fungicides containing trizole groupâ&#x20AC;&#x2122;s exposure and chromosome aberrations. The fungicide PCZ has great potential to alter the DNA model. Therefore, we should avoid the extensive use of these fungicides in field crop and its surface run-off at near water bodies.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

BIOLOGY

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Ecotoxicological bioassays of the earthworms Allolobophora caliginosa Savigny and Pheretima hawayana Rosa treated with arsenate under laboratory conditions Abdelmonem Mohamed Khalil Department of Zoology, Faculty of Science, Zagazig University, Egypt

ABSTRACT

Little research has been carried out on the effect of arsenate on earthworms. Ecotoxicological laboratory tests are fundamental tools for assessing the toxicity of arsenate to soil organisms. In this study, the impact of arsenate on the survival, reproduction and behaviour of the endogeic earthworms Allolobophora caliginosa and the anecic earthworms Pheretima hawayana has been quantified. The 96-h LC50 of arsenate was estimated as 233.43 mg arsenate kg -1 soil d.w. for P. hawayana which is significantly higher than that of A. caliginosa 1 47.24 ± 27.1 6 mg arsenate kg -1 soil d.w. The number of juveniles of P. hawayana was significantly higher than that of A. caliginosa at the arsenate concentrations 1 80, 240 and 400 mg kg -1 soil dry weight. With the exception of the control (6.5 mg arsenate kg -1 soil d.w.), P. hawayana showed an avoidance behaviour for soils treated with all tested concentrations.

A. caliginosa preferred soils treated with 6.5, 60, 11 0, 1 80 mg arsenate kg -1 soil d.w., while the avoidance behaviour has been recorded only at 240 and 400 mg arsenate kg -1 soil d.w. This means that A. caliginosa individuals feed less when exposed to arsenate. On the contrary, the P. hawayana worms could be escaped into their deep vertical burrows when exposed to arsenate.

Key words: Acute Toxicity; Earthworm; Chronic Toxicity; Avoidance; Ecotoxicology; Bioassays.

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Corresponding author:

Abdelmonem Mohamed Khalil Department of Zoology, Faculty of Science, Zagazig University, Egypt E-mail: abdulmonem111 @gmail.com Original Submission: 28 September 201 3; Revised Submission: 02 November 201 3; Accepted: 11 November 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, B261 -B268

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Khalil Earthworms Allolobophora caliginosa and Pheretima hawayana treated with arsenate

INTRODUCTION Large quantities of contaminants have continuously been leaked into ecosystems as a consequence of urbanization and industrial processes [1 ].

As defined by Cazes [2], heavy metal pollution is the case where the quantities of these elements in soils are higher than the natural environmental concentrations and is potentially harmful to biological life.

Arsenic is a metalloid pollutant widely introduced into nature and the environment through industrial processes and agricultural practices [3-6]. Arsenic has been ranked in abundance as 20 th in the earthâ&#x20AC;&#x2122;s crust, 1 4 th in seawater and 1 2 th in the human body [7]. There has been growing concern about the environmental effects resulting from arsenic compounds and, therefore, different countries have established quantify the risks to animals posed various arsenic concentration limits for the protection of wild life in all ecosystems. Arsenic exhibits both metallic and non-metallic properties. In general, for metallic forms, arsenite (As3) is considered more toxic than arsenate (As5) [8-9]. Since arsenic is a normal constituent of the environment, there is a need for effective monitoring and measurement of arsenic at arsenic-containing soil and waste sites.

Arsenic occurs naturally in elevated concentrations in soils as a result of the weathering of the parent rock [1 0]. Over 200 arsenic-containing minerals have been identified, with approximately 60% being arsenates, 20% sulphides and sulphosalts and the remaining 20% including arsenides, arsenites and oxides [11 -1 2]. However, levels of 1 00â&#x20AC;&#x201C;2500 mg arsenate kg -1 soil have been found in the vicinity of copper smelters [1 3].

Ecotoxicological laboratory tests are considered a preliminary step in bioassays of environmental risk. Because of their relatively quick results, these tests can quantify the risks to animals posed by the use of certain substances in the soils of a given terrestrial ecosystem. Toxicity studies on soil fauna have not yet been carried out for several heavy metals. Such tests are also required to implement rules that can effectively regulate agricultural protection products. Since earthworms are one of the most important biotic components in the soil, they have been utilized extensively in studies of the effects of heavy metals [1 4-24]. Earthworms are known to play a major role in the mixing of soil constituents and are more susceptible to metal pol-

lution than many other groups of terrestrial invertebrates [25]. An avoidance response test has been used in many studies to assess the toxicity of contaminated soils to earthworms [26-35]. Most studies have evaluated arsenic toxicity in mammal cell lines and aquatic animal species. However, the studies of arsenic toxicity in terrestrial invertebrates, including earthworms are relatively limited.

The aim of this study was to characterize the effects of varying concentrations of arsenate on survival, growth, reproduction and behaviour of two earthworm species belonging to two different ecological categories: endogeic (A. caliginosa) and anecic (P. hawayana) under laboratory conditions.

MATERIALS AND METHODS Soil

Samples of a sandy loam soil have been collected from an orange orchard that has not been treated with inorganic as for 40 years. Background arsenic concentration in soil (control) was determined according to the methods of Anderson et al. [36].

Chemicals

All reagents used were analytical grade and all aqueous solutions were prepared using distilled water. Inorganic sodium arsenate (Na 2HAsO 4â&#x2013;Ş 7H 2O) was purchased from Sigma-Aldrich, Egypt.

Experimental animals

Clitellated specimens of two earthworm species and P. hawayana were sampled in same duration in selected sites (non-polluted soil) by diggingsoil and were separated by hand sorting.

Samples of each species were taken at the time of each sampling and placed in ventilated plastic boxes with their native soils and some leaf letter.

The boxes containing earthworms and soil were returned to the laboratory in Faculty of Science, Zagazig University. The concentrations of arsenate used in the laboratory tests were determined through range finding tests. Experiments were started with an acute toxicity using increasing concentrations up to the limit of 400 mg kg -1 soil d.w. The chronic toxicity bioassays used sublethal concentrations based on the present definitive lethality tests. The avoidance tests used a different set of sublethal concentrations, lower than that used for chronic toxicity tests.

A. caliginosa

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Acute toxicity

Approximately 400 g of uncontaminated orchard soil were partially air dried, sieved (3 mm) and placed in circular plastic containers with a diameter 1 2.5 cm and a height of 9.5 cm such that each container contained soil to a depth 5-7 cm. The soil was then rewetted to a moisture content (55%) using distilled water (control) or a solution of sodium arsenate to give soil concentrations of 60, 11 0, 1 80, 240 and 400 mg arsenate kg -1 soil d. w. Three replicates of each concentration in addition to the control (untreated soil) were examined. The concentrations used were based on the results of pilot experiments (not reported here). Earthworms were washed and 20 individuals of each species were placed in each test container which was closed with plastic mesh. Lethal concentrations (96-h LC50) of A. caliginosa and P. hawayana were assessed by probit analysis [37-38]. After linearization of response curves by logarithmic transformation of concentrations, 95% confidence limits and slope function were calculated to provide a consistent presentation of toxicity data. The worms were fed cow manure each day during the 4 days of the test. In the last day of bioassays, worms were removed from the containers. Individuals that did not respond to the mechanical stimulation of the anterior portion of the body were recorded as dead.

Chronic toxicity

Chronic toxicity effects of arsenate on earthworm reproduction and growth were assessed according to protocol of ISO [39]. The bioassays were installed as in the acute toxicity tests which differed only in duration, concentrations and assessment methods. The soil was treated with sublethal concentrations 20, 30, 50, 1 00 and 1 50 mg arsenate kg -1 soil d. w., for A. caliginosa and P. hawayana.

Three replicates of each concentration in addition to the control were used for 50 days. These concentrations were chosen on the basis of the data of previous acute toxicity test. Earthworms were washed and weighed individually and 20 individuals of each species with 600 g native soil were placed in each test container which was closed with plastic mesh. In the 25 day of bioassay, worms were removed from the containers. Individuals that did not respond to the mechanical stimulation of the anterior portion of the body were recorded as dead. Live worms were washed and

weighed and the difference between starting and ending body weight was calculated. For the next 25 days only the soils, juvenile worms and cocoons remained in the containers. On the 50th day, the containers were immersed in warm water (40 ± 5°C) for 2 h and once the juveniles had emerged on the soil surface, the number of individuals generated was calculated. All worms were fed with cow manure once a week during time of experiment.

In the chronic toxicity, the mean body weight difference and number of juveniles were calculated.

Earthworm’s behaviour

Avoidance tests were carried out following the recommendations of Alves et al. [40]. For both A. caliginosa and P. hawayana, the arsenate was tested at concentrations 1 0, 20, 30, 40 and 50 mg kg -1 soil d. w. with 3 replicates for each concentration. Plastic boxes (20x1 5x1 0 cm) were divided into two equal compartments with a plastic divider.

To one compartment, 400 g of treated soil were added while the other compartment remained as a control (400 g of untreated soil). Immediately thereafter, the plastic divider was removed and 1 0 clitellated earthworms of each species were placed on the line separating the two compartments of the containers. Containers were covered with plastic mesh. No food was added during this experiment. After 48 h, the plastic dividers were inserted again and the soil in both compartments removed.

The number of earthworms present in each container (treated and control) were counted. Worms that were along the dividing line between the two compartments were counted as 0.5 for each compartment.

Percentage avoidance was calculated according to Amorim et al. [41 ] using the equation: % avoidance = [(C-T)/N] x 1 00 where C is the number of worms in control soil, T is the number of worms in treated soil and N is the total number of worms at the beginning of experiment. Positive percentages indicate avoidance of the treated soil, a zero indicates no avoidance and the negative percentages indicate an attraction for the arsenic-treated soil.

Statistical analysis

Analysis of data was carried out by student’s t-test for comparing the means of experimental and control groups.

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RESULTS

25 days exposure to all examined concentrations (Fig. 2a & b).

Acute toxicity

In the control (background as concentrations in uncontaminated soil were 6.5 ± 0.3 mg kg -1 soil d. w.) of the acute toxicity tests, mortality of adult A. caliginosa and P. hawayana was below 1 0%, achieving the requirements of bioassay validation.

There were no significant differences between replicates for each treatment, therefore the data from the three replicates were pooled for statistical analyses. The 96-h continuous exposure LC50 for P. hawayana was significantly higher (233.43 ± 42.45 mg arsenate kg -1 soil. d. w.) than that of A. caliginosa (1 47.24 ± 27.1 6 mg arsenate kg -1 soil d.w) (p<0.05). Surviving worms also had reduced body weight and in some cases, morphological abnormalities like, coiling and curling and lifting the body of A. caliginosa have been recorded.

Chronic toxicity

Avoidance responses

In avoidance tests, the number of dead and escaped worms was < 1 0%. This criterion was fulfilled in all tests. P. hawayana worms significantly (p< 0.05) avoided soils treated with all tested arsenate concentrations compared to the control (untreated). Avoidance percentages were 27, 37, 30, 43 and 40% at 60, 11 0, 1 80, 240 and 400 mg arsenate kg -1 soil d. w., respectively (Fig. 3a) Soil treated with arsenate attracted A. caliginosa worms at the concentrations 60, 11 0, 1 80 mg kg -1 soil d. w.

However, soil treated with higher concentrations of the arsenate (240 and 400 mg kg -1 soil d. w.) was significantly (p<0.05) avoided by the worms (Fig. 3b).

DISCUSSION

In the chronic toxicity tests, the control showed means of 75 and 92 juveniles of A. caliginosa and P. hawayana, respectively. The number of produced A. calignosa juveniles were significantly (p<0.05) fewer than that of P. hawayana at the concentrations 1 80, 240 and 400 mg arsenate kg -1 soil d.w.

Number of juveniles of P. hawayana were four times larger (40 individuals) than the number of juveniles of A. caliginosa (1 0 individuals) at the concentration 400 mg arsenate kg -1 soil d.w. (Fig. 1 ). Examined P. hawayana showed significant (p< 0.05) reduction in body weight after 25 days exposure to concentration 60 mg arsenate kg -1 soil d.w. compared to the control while A. caliginosa individuals showed significant (p<0.05) reduction in body weight after

Earthworms are key organisms in soil ecology.

As ecosystem engineers their activity affects not only many important soil processes such as soil

Fig. 1. Mean A. caliginosa

Fig. 2.

number of juveniles of P. hawayana and treated with varying concentrations of

arsenate. * Means differ significantly from t-test, P < 0.05).

A. caliginosa

(Student’s

Reduction in biomass (mg) of (a): P. hawayana and (b): A. caliginosa treated with varying concentrations of arsenate (25 days of exposure). * Means differ significantly from control (Student’s t-test, P < 0.05).

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Khalil Earthworms Allolobophora caliginosa and Pheretima hawayana treated with arsenate A

B

Fig. 3. Avoidance or attraction response of (a): P. hawa­ yana and (b): A. calignosa to arsenate concentrations

(mean net response and standard error bars). * Means differ significantly from control (student’s t-test, P < 0.05).

aeration, decomposition or nutrient availability, but they also promote soil biodiversity, soil fertility and soil health [42]. In this study both acute and chronic toxicity effects of arsenate have been demonstrated. The 96-h LC50 (233.43 ± 42.45 mg kg -1 soil d. w.) of P. hawayana was significantly (p< 0.05) higher than that of A. caliginosa (1 47.24 ± 27.1 6 mg kg -1 soil d. w.). The difference between the acute toxicity of the two worms could be explained on the basis of the variety of their living and feeding habitats. A. caliginosa is an endogeic earthworm and the individuals of this species forage below the surface where they ingest large quantities of organic-rich soil and build burrows that are mainly horizontal in nature. Although they build permanent and vertical burrows that penetrate the soil deeply, the individuals of anceic species P. hawayana come to the surface to feed on partially decomposed litter,

manure and other organic matter. Their burrows create microclimatic gradient and the worms can be found shallow or deep in their burrows depending on the prevailing conditions. The differences between the results reported in the literature and those of the present study are small and mostly attributable to different substrate composition and the earthworm species [36]. Body weight loss may reflect reduced feeding by the worms as reported in other studies [43-44]. In the present study, the body weight of A. caliginosa treated with arsenate concentrations (11 0-400 mg kg -1 soil d. w.) significantly (p<0.05) reduced in relation to the control while the body weight loss in p.hawayana was not affected by the arsenate concentrations except at 60 mg kg -1 soil d. w. This means that A. caliginosa individuals feed less when exposed to arsenate.

On the contrary, the anecic P. hawayana worms could be escaped into their deep vertical burrows when exposed to arsenate. However, Kreutzweiser et al. [45] noted that worm feeding was similar in treated and control soils and attributed the reduction in biomass to physiological changes in worms. Luo et al. [46] shown that a reduction of cellulase activity compromises the feeding efficiency of exposed Eisenia fetida, resulting in lower weight gain. Buffun [47] introduced another possible explanation for the lethal effect involves a blocking of nervous system receptors by pesticides. This blocking leads to an accumulation of acetylcholine which results in muscle and organ paralysis and depend on its intensity, can kill earthworms [48]. The lethal effects could be attributed to the blocking of nervous system receptors by pesticides. In P. hawayana, the lowest concentration of arsenate (60 mg kg -1 soil d. w.) resulted in significant (p<0.05) less weight loss than the control (Fig. 3b) which could be the result of physiological stimuli caused by low concentrations of arsenate. Some authors have reported similar results and attributed it to a hormetic response which is defined as the ability of a substance to be toxic at high concentrations but stimulant at low concentrations [49]. Few number of researches have been performed on the impact of heavy metals on earthworms avoidance under laboratory conditions. The earth worm avoidance test, originally developed in USA [50], was selected because it is quick and easy to perform and it is known to be sensitive towards a wide range of chemicals. Natal-du-luz et al. [51 ] conducted avoidance tests with earthworms to demonstrate their feasibili-

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Khalil Earthworms Allolobophora caliginosa and Pheretima hawayana treated with arsenate ty as early screening tools for assessing the toxic potential of metal-polluted soils. Resuts as abridged in Fig. 3a & b, it could be demonstrated that avoidance responses of P. hawayana is more sensitive to arsenate contamination than that of A. caliginosa.

In this study, avoidance response of A. caliginosa occurred at a concentration 4 times (240 mg kg -1 soil d. w.) higher than that of P. hawayana (60 mg kg -1 soil d. w.). The ability of A. caliginosa to tolerate high levels of arsenate probably involves a combination of heritable and integrated physiological, morphological and behavioral modifications. Increased bio-immobilization capacity may be the most likely physiological strategy for arsenate resistance in earthworms [1 5].

Based on the results of this work, it is concluded that body weight loss may reflect reduced feeding by the worms. The body weight of A. caliginosa treated with arsenate significantly reduced in relation to the control while the body weight loss in P. hawayana was not affected by arsenate concentration except at 60 mg kg -1 soil d. w. For assessing the toxicity of arsenate, A. caliginosa and P. hawayana are ecologically relevant species and they are sensitive to arsenate applications. However, when a rapid and efficient screening is required, P. hawayana is much more convenient.

TRANSPARENCY DECLARATION

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The authors declare no conflicts of interest.

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Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Insecticidal effect of methoprene on the pre-adult stages of almond moth, Ephestia cautella Walker (Lepidoptera: Pyralidae) Awanish Chandra, Shri Krishna Tiwari Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur, India

ABSTRACT

The larvicidal and pupicidal effects of methoprene were studied on the third-instar larvae of almond moth, Ephestia cautella Walker. The almond moth, E. cautella is a serious pest of stored cereals and cereal products. Methoprene is a safe alternative to conventional insecticide. In presented studies was demonstrated, that methoprene causes a significant enhancement in the larval mortality and a significant reduction in pupation and adult emergence. At 1 ppm of methoprene, larval mortality was 11 .80 ± 1 .70% that increased to 1 00% at 1 4 ppm. At 1 2 ppm of methoprene, 1 5.00 ± 3.03% larvae pupate but all get perished and hence none of them emerge. The findings suggest that methoprene adversely affects the developmental stages of Ephestia cautella Walker.

Key words: Stored cereal pest; Larvae; Ontogeny; Insecticide; Mortality.

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Corresponding author:

Shri Krishna Tiwari Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur - 273009 (U.P.) India E-mail: sktzddu@rediffmail.com Original Submission: 1 9 October 201 3; Revised Submission: 27 November 201 3; Accepted: 02 December 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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INTRODUCTION Insect infestation in stored products causes serious damage to the infested commodity. Post-harvest losses in India amount to 1 2 to 1 6 million metric tons of food grains each year, an amount that the World Bank estimates could feed one-third of India's poor [1 ]. The monetary value of these losses amounts to more than Rs. 50,000 crores per year [2]. During storage, quantitative as well as qualitative losses occur due to insects, rodents, and microorganisms. A large number of insect pests have been reported to be associated with stored grains.

Preventive measures utilizing conventional grain protectants (organic insecticides) may result in the presence of chemical residues [3] in foodstuffs that may eventually be destined for human consumption. There is an evidence of increasing incidence of resistance to conventional insecticides among the major insect pests of stored products. In addition, their persistent use affects immune system of insects and of course pollutes our own environment due to nonbiodegradability, biomagnifications and toxicity to non-target organisms. In such condition safer substitutes are the need of the present environment. Williams [4] coined the term “third generation pesticides” to describe hormone-based insect growth regulators (IGRs). There has been a renewed interest in IGRs usage, specifically in the capacity as grain protectant treatment, surface treatments, as well as aerosol and fogging treatments in the interior of food storage structures [5].

The almond moth, E. cautella is a serious pest of stored cereals and cereal products in India, Indonesia, South America, Turkey and both tropical and temperate region of the world [6-8]. Its larval stages cause serious damage to wheat, maize, cocoa beans, dried fruits, ground nuts, pea nuts, stored vegetables, coffee beans, oil cakes and chocolate products [7-11 ]. This moth not only causes loss in weight, but the presence of larvae also cause considerable damage by contaminating stored food with dead bodies and their own products e.g. excreta, webbing, silk and faeces [11 , 1 2]. Synthetic insecticides, both as contact insecticides and fumigants, have been used extensively to control stored-product insects since the 1 960’s [1 3, 1 4].

However, at present there is an emphasis on the use of insecticides that have biorational properties [1 5] owing to many concerns about the use of currently available insecticides [1 3]. IGRs with juvenile

hormone activity, otherwise known as juvenile hormone analogues (JHA), are nonpoisonous and do not bioaccumulate, therefore they generally do not persist for prolonged periods in the environment.

IGRs have been shown to generally have a good margin of safety for most non-target biota, as they display a very low toxicity for humans and other mammals, are readily biodegradable (i.e., very low persistence in the environment), highly toxic to target insects, and leave no hazardous residues, making JHAs very useful in food preservation and storage [1 6]. The objective of the present study is to assess whether the methoprene is adequately effective or not in case of E. cautella. Hence, as an objective of such programme the present study, for the first time, has been designed and conducted to investigate the influence of methoprene, a juvenile hormone analogue, against the ontogeny of almond moth, E. cautella. Such knowledge may be regarded as one of the objective criteria permitting an assessment of effectiveness of hormonal control measures against E. cautella in particular and lepidopterous pests in general.

MATERIALS AND METHODS The almond moth, Ephestia cautella Walker was collected from the Central and State Warehouse Corporations located at Gorakhpur, U.P., India.

A rich standard culture of this insect was maintained in the laboratory on a normal dietary medium composed of coarsely ground wheat (Triticum aeste­ vum) mixed with 5% (w/w) yeast powder and 1 0% (w/w) glucose inside large glass containers (1 50 mm diameter, 200 mm height) at a temperature of 26 ± 1 0ºC, relative humidity 93 ± 5% and a light regime of 1 2 hours light and 1 2 hours darkness. From the above culture whenever needed, newly emerged males and females were transferred to oviposition glass chambers (35 mm diameter, 200 mm height). Since, E. cautella Walker individuals do not feed during their adult stage, no food was provided to them during their confinement in these vessels.

Eggs laid by the females were collected and then placed in glass chambers (consisting of 250 ml beakers) for hatching.

Methoprene (Isopropyl (2E,4E)-11 -methoxy-3,7, 11 -trimethyl-2-1 , 4-dodecadienoate), molecular formula: C 1 9H 34O 3 (7.4% cis and 90.4% trans), 97.8% (a.i.) was used throughout the investigation and was obtained from AccuStandard, Inc.1 25 Market Street,

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For the preparation of different ppm cocentrations of methoprene in dietary media, a stock solution of known concentration of methoprene was prepared in required acetone and then adjusted via serial dilutions to achieve its required concentrations. Now required volume of different concentrations of methoprene were thoroughly mixed with the required quantity of normal food (coarsely ground wheat, Triticum aestivum mixed with 5% (w/w) yeast powder and 1 0% (w/w) glucose) to get desired ppm levels of methoprene. This treated food was then air dried at room temperature to eliminate completely the excess of the organic solvent. For control purposes, the normal food was thoroughly mixed with a required volume of acetone similar to that of treated food and then air dried in the same way.

To evaluate the toxic effects of various concentrations of methoprene, freshly hatched larvae of E. cautella were allowed to feed on a normal dietary medium (kept inside 250 ml beakers) for exactly 20 days. On the 21 st day, 25 third instar larvae were transferred to each similar rearing chamber containing 50 gms of dietary medium mixed and treated separately with different known concentration levels of methoprene. Experiments were conducted on 8 different concentrations of methoprene i. e. 1 , 2, 4, 6, 8, 1 0, 1 2 and 1 4 ppm. 25 larvae were also kept on a normal dietary medium as control.

On the completion of developmental cycle, percent Table 1. Toxicity

adult emergence and percent pupal mortality was observed and on that basis percent pupation and percent larval mortality was calculated. The corrected total larval mortality was calculated by Abbott’s formula [1 7]: Corrected larval mortality = 1 00 x [(% experimental mortality – % control mortality) / (1 00 – % control mortality)] Experiments were replicated six times and the values have been expressed as the mean ± S.D. Straight line regression equation was applied between different concentrations of juvenile hormone analogue and their corresponding percent larval mortality/percent pupation/percent pupal mortality and percent adult emergence to observe the significant correlation.

RESULTS AND DISCUSSION Table 1 represents toxicodynamic property of methoprene on the ontogeny of E. cautella Walker.

A significant larval and an insignificant pupal mortality was obtained with the increases of methoprene concentration in the diet. At 1 ppm level, larval mortality was 11 .80 ± 1 .70% while 1 00% larval mortality was recorded at 1 4 ppm. As the methoprene concentration increase a significant reduction in pupation and an insignificant enhancement in pupal mortality occur. At 1 ppm of this insecticide, 84.67 ± 1 .63% pupation was recorded which decre-

of methoprene against the ontogeny of almond moth,

Ephestia cauttella

Walker.

*Values have been expressed as the mean ± SD of six replicates.

Straight line regression equation was applied between different dose levels of methoprene and their corresponding percent larval mortality/pupation/pupal mortality/adult emergence for significant correlation.

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Chandra & Tiwari Insecticidal effect of methoprene on almond moth Ephestia cautellae ased to 1 5.00 ± 3.03% at 1 2 ppm methoprene. At the same time, 1 8.68 ± 2.89% pupal mortality was recorded at 2 ppm, which insignificantly increased to 1 00% at 1 2 ppm. At 1 2 ppm of the test insecticide 84.37 ± 3.1 4% larval mortality occurred and therefore 1 5 ± 3.03% larvae pupated and all perished. It deserves mention that at 1 ppm there was no pupal mortality. A significant reduction in adult emergence was recorded following exposure of increased concentrations of methoprene. At 1 ppm methoprene 84.67 ± 4.68% adult emergence was recorded that decreased to 2.67 ± 2.06% at its 1 0 ppm level. It was also observed that low concentration levels of methoprene extended the larval life span poorly but higher levels i.e. 1 4 ppm of methoprene extended the larval tenure near about two weeks and then after the larvae turned black, shrinked into solid tissue mass and thereafter died.

The present investigation, for the first time, reveals several essential and interesting information concerning some of the so far unexplored methoprene induced changes on the life-cycle stages of almond moth, E. cautella Walker. It deserves mention that in the present investigation the highest level of methoprene (1 4 ppm) that caused 1 00% larval mortality, the larval food consumption was observed to be very poor. This observation indicates that at this highest concentration level the larval tenure extended about two weeks in comparison to the larvae treated with the low levels of methoprene. Observation revealed that the highest concentration of methoprene inhibited metamorphosis that led to 1 00% larval mortality. Juvenile hormone is involved in maintaining the diapauses in some species with larval diapauses such as Diatraea grandiosella Dyar [1 8]. High methoprene levels during larval stage disrupt development of the E. cautella. This study indicates that methoprene may disrupt the normal development of this insect.

At low level of methoprene it would be interesting to determine if larvae that survive methoprene exposure and adult emergence occurred, maximum adults were abnormal. There are several examples that insects have a window of sensitivity to JH or JHAs. Larvae are sensitive and adults are not sensitive to JH and JHA [1 9]. Failure to emerge as an adult of Tribolium castaneum due to methoprene treatment is well documented [1 9, 20]. In insects with complete metamorphosis, JH titers generally remain low in the later part of the last larval instar and the pupal stage for normal deve-

lopment of insects [21 ]. High JHA levels during immature stages disrupt development of the insects [22]. Within the egg and larval stages also, there are examples of JH-sensitive phases. With Oncopeltus fasciatus, JH must be present during the fourth instar larvae for the normal development of accessory glands, and the JH deficiency during that period cannot be recovered by having JH at a later stage [23]. Alternatively, juvenile hormone levels may be high in the adult [24, 25], and external application of methoprene may not be enough to disrupt the reproductive systems.

Loschiavo [26] examined that 20 ppm of methoprene and hydroprene prevented pupation of Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) and substantially reduced the adult emergence in T. confusium. Larvae that failed to pupate in treated food contained to molt and those survive to 1 20 days or longer after emergence were larger than normal larvae. Loschiavo [27] reported at 1 0 and 20 ppm of methoprene against of rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae) caused 1 0 and 61 % progeny suppression respectively. Strong and Diekman [28] noted that at 5 ppm Altosid (methoprene) no progeny suppression of rice weevil, S. oryzae (L.) on wheat and 7 and 92% suppression respectively. However, Mian and Mulla [29] achieved at 1 0 ppm of methoprene again S. oryzae the progeny suppression was 80.7-93.1 % in wheat. Arthur [30] achieved that 1 0 ppm of R, S-methoprene (a racemic mixture of the R and S isomers of methoprene) gave 1 00% suppression of F1 adult progeny of Rhyzopertha dominica (Coleoptera: Bostrichidae), Chanbang et al. [31 ] reported that the larvae of R. dominica can developed from eggs exposed to methoprene and can bore into the kernels, but do not emerge as adults and Athanassiou et al. [32] noted that at 5 and 1 0 ppm of methoprene were effective against R. dominica on wheat, rice and maize as the whole grain treatment. Shaaya et al. [33] found that methoprene interfered with the development of eggs and larvae of E. cautella in an age-dependent manner, and showed that only certain stages were susceptible. Metamorphosis was inhibited and supernumerary larvae were produced. Our findings resemble to that of Oberlander and Silhacek [1 9]; Retnakaran et al. [20] and Athanassiou et al. [32].

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ACKNOWLEDGEMENTS The authors thank Prof. V.B. Upadhyay, Head, Department of Zoology, D.D.U. Gorakhpur University Gorakhpur for providing laboratory facilities, to UGC, New Delhi for financial assistance F1 1 7.1 /2011 -1 2/ RGNF-SC-UTT-4607/ (SA-III/ Website) dated 06-June-201 2 to first author and to AccuStandard, Inc.1 25 Market Street, New Haven for providing methoprene. Thanks are also due to Zoological survey of India, Kolkata for identification of the present insect (F. No. ENT/225/1 0490 dated 08-Sept-2011 ).

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Gibberellic acid ameliorates the adverse effects of acid mist and improved antioxidant defense, water status and growth of acid misted sunflower plants Suzan A. Sayed, Mohamed A.A. Gadallah Botany Department, Faculty of Science, Assiut University, Assiut, Egypt

ABSTRACT

Pot experiment was conducted to determine the effect of gibberellic acid (GA3) application on sunflower (Helianthus annuus) plants exposed to acid mist of pH 2.0. Sunflower plants were sprayed with HNO 3, HCl and H 2SO 4 acid solutions of pH 2.0 or distilled water as control and then sprayed with 0 and 1 00 mgL-1 gibberellic acid solution. In the absence of GA3 plants sprayed with HCl and H 2SO 4 solutions had lower contents of chlorophyll (Chl), soluble sugars (SS) and soluble proteins (SP) and produced less biomass than the unacidified control. The reverse held true in plants received HNO 3 solution. Membrane injury by dehydration (40% PEG) and heat stress (51 ºC), hydrogen peroxide (H 2O 2), lipid peroxidation (LP) and proline contents were increased with exposure to HCl acid mist of pH 2.0. Gibberellic acid treatment counteracted to variable extents the adverse effects of acid mist on the above physiological parameters. Effectively it is protecting the plant membrane from dehydration and heat stress injury, decreasing H 2O 2 and lipid perioxdation levels and enhancing ascorbic acid production and growth.

These finding suggest that GA3 treatment can ameliorate the negative effect of the acid mist and improved antioxidant defense and water status of acid misted sunflower plants.

Key words: Acid mist; Ascorbic acid; Helianthus annuus; Hydrogen peroxide; Lipid peroxidation; Membrane stability.

J Biol Earth Sci 201 3; 3(2): B275-B285

Corresponding author:

Suzan A. Sayed Botany Department, Faculty of Science, Assiut University, 71 51 6 Assiut, Egypt Fax: 0020882342708 Tel. 00201 09801 9725 E-mail: drsuzan1 @hotmail.com Original Submission: 1 9 October 201 3; Revised Submission: 27 November 201 3; Accepted: 02 December 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abbreviations: AM = acid mist, Asc A = ascorbic acid, Chl = chlorophyll, GA3 = gibberellic acid, LP = lipid pero-xidation, MDA = malondialdehyde, RWC = relative water content, SS = soluble sugars, SP = soluble proteins, PEG = polyethylene glycol, TAA = total free amino acids, TCA = trichloroacetic acid.

INTRODUCTION Burning of fossil fuels in industries and transport sector, industrialization and urbanization have led to increase in concentrations of gaseous and particulate pollutants in the atmosphere leading to air pollution [1 -3]. Acid rain is currently regarded as one of the most widespread and environmentally significant consequence of atmospheric pollution, rapid industrialization, urbanization and development.

The acidity of rain in many parts of the world has increased over the past years and this phenomenon has been linked primarily to increased emission of oxides of sulphur and nitrogen from the combustion of fossil fuels [4].

The effect of continued acid rain exposure on the economic crops is of considerable interest. Crop plants showed a wide range of sensitivity to the acidity of rain. Acid rain induces changes in the cellular biochemistry and physiology of the whole plants.

Biological effects of acid deposition on plants are numerous and complex and include visible symptoms of injury (chlorosis and /or necrosis) and invisible effects such as reduced photosynthesis, nutrient loss from leaves, altered water balance and variation of several enzymes activities [5-1 0].

Plants operate several mechanisms to counteract the adverse effects of air pollution. These mechanisms may be enhanced by the application of chemicals to plants. One of such mechanism is the activation of an antioxidant enzyme system, which may be influences by the interaction of plant growth regulators and acid rain [7].

Plant hormones are active members of the signal cascade involved in the induction of plant stress response [11 , 1 2]. Exogenous application of growth hormones may be useful to return metabolic activities to their normal levels. Gibberellins (GAs) are plant growth regulators that are known to stimulate physiological response in plants and alter the source-sink metabolism through their effect on photosynthesis and sink formation. Recent studies indicate that GA signaling is involved in adjustment of plants under limiting environmental conditions

[1 3]. The role of AG 3 in the regulation of plant responses to a biotic stress has been documented [1 4-1 8].

Scare data are available on the effect of exogenous gibberellic acid and acid mist treatment in combination. Often their effects on the growth and metabolic activities of the plants have been studied separately (as single factorial treatment). Accordingly, in the present paper, the possibility of acid mist and gibberellic acid interaction affecting chlorophyll content, dry mass production as well as some antioxidant parameters and soluble carbon and nitrogen fractions were studied. Since damage from acid rain takes place firstly on the plant surface [7, 1 9] and since the extent of membrane damage by adverse environmental condition such as drought, heat, salinity and waterlogging is used as a measure of stress tolerance [20], the degree of membrane injury by acid rain could be a good measure for plant tolerance to acid rain. Therefore, this study was undertaken also to investigate the combined effects of an acid spray of pH 2.0 and gibberellic acid on the stability of leaf membrane of sunflower plants.

MATERIALS AND METHODS Plant material and treatments

Sunflower (Helianthus annuus L. var. macrocar­ (DC.) Cockerell) were grown in plastic pots (5 kg capacity) in the Experimental outdoor green house at the Faculty of Science, Assiut University (Egypt) under natural field conditions. The minimum and maximum air temperature and relative humidity were 22 and 38ºC and 45 and 60%, respectively.

The average photoperiod during the experiment was 1 2 h. After 3 weeks the growing plants (three per pot) were either sprayed (three times at 2 day intervals) with distilled water (control) or distilled water acidified with hydrochloric acid (HCl), nitric acid (HNO 3) or sulphuric acid (H 2SO 4) to achieve pH value of 2.0. Misted or unmisted plants were then sprayed with 0 and 1 00 mgL-1 gibberellic acid solutions. Spraying was done once a day for one week. Five pots were assigned at random to each treatment combination. Treated plants were analyzed 4 days after the last gibberellic acid application. pus

Membrane stability

The stability of leaf membranes was assessed by determining leakage of electrolytes from leaf di-

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Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants scs exposed to dehydration (40% polyethylene glycol) and heat (51 °C) stress. A method used by Blum and Ebercon [20] on wheat. According to Gadallah [7], leaf discs of 1 0 mm in diameter were punched out of the fully-expanded leaves and washed with de-ionized water to remove surface electrolytes.

Five discs of each treatment were placed in each of (a) wetted test tubes and heated to 51 °C for 20 min in a water bath then incubated for 20 h at 1 0°C (heat test), (b) test tubes with 1 0 ml de-ionized water (controls) and (c) test tubes with 1 0 ml 40% PEG 6000 (drought test) and incubated for 20 h at 1 0°C. All samples (leaf discs in a, b and c) were washed three times with de-ionized and re-incubated with 1 0 ml de-ionized water for further 20 hr at 1 0°C. The electrical conductance was measured at 25°C after equilibration of samples in water bath using conduct-meter YSI Model 35. Following autoclaving at 1 00°C for 1 5 min and re-equilibration at 25°C, the conductance was measured a second time.

The degree of membrane injury (based on electrical conductance measurements) was calculated according to the following formula: % injury = 1 - [1 - (T1 /T2)/1 - (C1 /C2)] x 1 00 where T1 and T2 represent the first and second measurements on the treatments samples and C1 and C2 the first and second measurements on the control.

Chlorophyll determination

The pigment fractions (chlorophyll a and chlorophyll b) were estimated in 95% ethanol extracts at 60ºC (Extracts centrifuged at 3,000×g and absorbance was recorded at 646.8 nm and 663.2 nm) following the spectrophotometric method (Unico UV21 00 spectrophotometer) recommended by Lichtenthaler [21 ].

Leaf relative water content

The leaf relative water content (RWC) was calculated from fresh, turgid and dry weight of leaf discs, as described by Silveira and other [22]. Fresh leaf samples were taken from fully expanded leaves and weighed (FW), after which the samples were immediately hydrated to full turgidity for 4h under normal room light and temperature. After 4h samples were taken out of water and were well dried of any surface moisture quickly and lightly with filter paper and immediately weighed to obtain fully turgid weight (TW). Samples are then oven dried at 80°C for 24h and weighed (after being cooled down in a

desiccator) to determine dry weight (DW).

Calculation: L RWC (%) = (FW - DW/TW - DW) × 1 00

Determination of the malonydialdhyde (MDA)

The level of lipid peroxidation in plant tissues was measured by determination of MDA [23, 24].

MDA content was determined with thiobarbituric acid (TBA) reaction. 0.2 g tissue sample was homogenized in 5 ml 0.1 % TCA. The homogenate was centrifuged at 1 0,000×g for 5 min. 4 ml of 20% TCA containing 0.5% TBA was added to 1 ml aliquot of the supernatant. The mixture was heated at 95°C for 1 5 min and cooled immediately in an ice bath.

The non-specific absorbance of the supernatant at 600 nm was subtracted from the maximum absorbance at 532 nm for MDA measurement. The level of lipid peroxidation was expressed as µmol of MDA formed using an extinction coefficient of 1 55 mM -1 cm -1 .

Determination of H 2O2

The H 2O 2 content of leaf samples was colorimetrically measured as described by Mukherjee and Choudhuri [25]. Leaf samples were extracted with cold acetone to determine H 2O 2 levels. An aliquot (1 mL) of the extracted solution was mixed with 200 mL of 0.1 % titanium dioxide in 20% (v/v) H 2SO 4, and the mixture was then centrifuged at 6,000 g for 1 5 min. The intensity of the yellow color of the supernatant was measured at 41 5 nm. The H 2O 2 concentration was calculated from a standard curve.

Determination of ascorbic acid

Ascorbic acid was estimated by the method of Jagota and Dani [26] by mixing 2 mol/L Folin-ciocalteu reagent and 1 0% TCA with 20% tissue homogenate. The blue colour developed in the supernatant after centrifugation was read at 760 nm after 1 0 min. The amount of ascorbic acid was calculated from a standard curve of vitamin C using 5–70 µg serial concentrations of the vitamin.

Dry matter determination

At the end of the experiment, three randomly chosen shoots were cut just above soil surface, washed in detergent solution to remove dust on leaf surfaces and then dried at 70°C for 48h to constant weight.

Soluble carbon and nitrogen metabolites

The contents of soluble sugars, free amino acids

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Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants and soluble proteins were determined in dry mass extracts according to the methods of Buysse and Merckx [27], Lee and Takahshi [28] and Lowry and other [29]; respectively. Calibration curves using glucose (soluble sugars), glycine (amino acids), and bovine serum albumin (soluble proteins) were constructed.

Statistical analysis

Statistical inferences necessary to evaluate the effects acid mist (pH), gibberellic acid (GA3) and their interaction (pH × GA3) on the parameters tested included: analysis of variance (ANOVA) and coefficient of determination (η 2) respectively [30].

RESULTS Membrane stability

Exposure of leaf discs excised from control plants to dehydration stress (40% PEG) caused 1 7.81 % injury (Fig. 1 ). The percent of injury in plants misted with HNO 3, H 2SO 4 and HCl solutions of pH 2.0 increased to 31 .94, 38.1 3 and 31 .26%, respectively. Heat stress (51 °C) caused about 35.72% in the membrane of unmisted plants. Leaf discs of plants received H 2SO 4 and HCl acid mist were injured more by heat stress than those sprayed with distilled water. On the contrary leaf membrane of plants received HNO 3 solution with pH 2.0 less injured (7.63) by PEG than unmisted control.

Gibberellic acid treatments protected leaf membranes from dehydration and heat stress damage, effectively it reduced membrane injury percent by either dehydration or heat stress compared with control not received gibberellic acid.

The effects of single factors, acid mist (pH), gibberellic acid (GA3) and their interaction (pH × GA3) had significant effect (Table 1 ) on the stability of leaf membranes to heat stress. The same held true for the effect of GA3 only on membrane stability to dehydration stress but the effects of acid (pH) and the interaction were statistically non significant. Calculation of the coefficient of determination (η 2) indicated that the role of GA3 in affecting membrane stability was dominant but the role of pH was subdominant.

Soluble protein and total free amino acids

Shoots of plants misted with the three acid solution of pH 2.0 showed lower content of soluble proteins than unmisted plant (Fig. 2). This was not the

case however with total free amino acid where their contents were slightly increased in shoot of HNO 3 and HCl acid misted plants but were reduced by H 2SO 4 solutions. Gibberellic acid application enhanced accumulation of soluble proteins as well as total free amino acids in sunflower plants whether misted or not. Total free amino acids of plants misted with acid solution of pH 2.0 and sprayed with GA3 were greatly higher than those not receiving GA3. The reverse held true in unmisted plants where the total free amino acids showed greater decrease in response to GA3 treatments.

Acid pH and GA3 as well as their interaction (Table 3), had significant effects on the contents of total free amino acids. However, soluble protein was significantly affected by GA3 only. The role of pH was dominant for total free amino acid while the role of GA3 was subdominant. The reverse held

Effects of acid mist of pH 2.0 and gibberellic acid (GA3, 1 00 mg L-1 ) sprays on the stability of leaf membranes (measured as % of membrane injury) to (A) heat stress (51 ºC) and (B) dehydration (40% PEG), (C) chlorophyll (Chl a) and (D) chlorophyll (Chl b) contents in sunflower plants. Data are means of five replicates ± SE. Fig. 1.

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Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants true for soluble proteins.

Soluble sugars

Plants misted with H 2SO 4 and HCl solutions (Fig. 2) showed greater decrease in the content of SS than plants unmisted. On the contrary, plants sprayed with HNO 3 accumulated more SS in their shoots than the unacidified plants. Gibberellic acid application enhanced SS accumulation in shoots of acid misted and unmisted plants as well. Data of Table1 indicate that soluble sugars content significantly affected by single factors (GA3) as well as bifactorial interaction. Based on the coefficient of determination (η 2), the role of GA3 role in affecting SS content was dominant followed by pH × GA3 interaction as subdominant but the effect of pH was minor one.

Effects of acid mist of pH 2.0 and gibberellic acid (GA3, 1 00mg L-1 ) sprays on the contents of (A) soluble proteins (SP), (B) total amino acids (TAA), (C) soluble sugars (SS) and (D) dry mass (DW) production in sunflower plants. Data are means of five replicates ± SE. Fig. 2.

Growth

Shoot dry mass production (Fig. 2) was decreased by H 2SO 4 solution of pH 2.0 but increased by either HNO 3 or HCl solutions. Generally GA3 application enhanced shoot dry mass production of acid misted and unmisted plants as well. The enhancement was more pronounced in acid misted plants. Shoot growth was significantly affected

by GA3 treatments but the effects of pH and pH × GA3 interaction were statistically non significant (Table 1 ). The role of GA3 in affecting shoot growth was dominant.

Chlorophyll content

Unmisted plants (Fig. 1 ) had higher contents of chlorophyll a and chlorophyll b than those sprayed with HCl and H 2SO 4 acid solutions. Contrarily,

Effects of acid mist of pH 2.0 and gibberellic acid (GA3, 1 00mg L-1 ) sprays on the contents of (A) ascorbic acid (Asc A), (B) malonyldialdehyde (MDA), (C) Hydrogen peroxide and (D) leaf relative water content (RWC) in sunflower plants. Data are means of five replicates ± SE. Fig 3.

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* - significant at 5% of probability levels, ** - significant at 1 % of probability levels; ns: Non-significant.

Table 2.

F and η 2 values for the effects of acid mist (pH), gibberellic acid (GA3)and their interaction (pH × GA3) on the contents of chlorophyll (Chl), ascorbic acid (Asc A), lipid peroxidation (MDA), hydrogen peroxide (H 2O 2) and leaf relative water content (RWC) of sunflower plants.

* - significant at 5% of probability levels, ** - significant at 1 % of probability levels; ns: Non-significant.

F and η 2 values for the effects of acid mist (pH), gibberellic acid (GA3) and their interaction (pH× GA3) on the stability of leaf membranes and contents of soluble proteins, total amino acid, soluble sugars and growth of sunflower plants.

Table 1.

Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants

B280

Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants plants receiving HNO 3 acid solution had higher chlorophyll a and b contents than those sprayed with distilled water. Gibberellic acid treatment increased significantly the content of chlorophyll a and b in acid misted plants. Chlorophyll increased by GA3 was more pronounced in plants sprayed with H 2SO 4 where Chl content was about two-fold that of plants not treated with gibberellic acid.

Statistical analysis (Table 2) show that pH, GA3 and their interaction significantly affected the contents of Chl as indicated by F value (pH × GA3 interaction was an exception for Chl a). Based on calculated coefficient of determination (η 2), GA3 had a dominant role in affecting Chl content while the role of pH and pH × GA3 interaction was subdominant.

Lipid peroxidation, hydrogen peroxide and ascorbic acids

Sunflower plants sprayed with an H 2SO 4 solution (Fig. 3) showed a greater decreased in content of H 2O 2 than those sprayed with water. The opposite response was noticed in plants sprayed with an either HNO 3 or HCl solutions. Plants receiving GA3, whether acidified or not, had lower contents of H 2O 2 than untreated analogues.

Plant misted with acid solution of pH 2.0 accumulated more MDA in their shoots than unacidified plants. Generally GA3 application reduced accumulation of MDA in sunflower plants misted by HNO 3 and HCl solutions as well as in unacidified plants but enhanced MDA accumulation in plants sprayed with H 2SO 4 solution.

Sunflower plants accumulated low amount of ascorbic acid in response to acid spraying. Plants which were exposed to HNO 3 and H 2SO 4 acidified solutions and received GA3 showed an increase in their content of ascorbic acid. On the contrary, GA3 reduced ascorbic acid content in unmisted and HCl misted plants.

Calculations in table 2 indicate that the contents of H 2O 2, MDA and ascorbic acid were significantly affected by acid pH and pH × GA3 interaction. Contrarily, the effect of GA3 on ascorbic acid and MDA was non significant. The role of pH in affecting ascorbic acid, and LP was dominant followed by pH× GA3 interaction (Asc acid) or GA3 as a subdominant. In case of H 2O 2, GA3 had dominant role followed by pH × GA3 interaction but the role of pH was minor one.

Leaf relative water content

Leaf RWC (Fig. 3) slightly affected by the three acid solutions. Mostly non significant decrease (with HNO 3 and HCl solutions) and increase (with H 2SO 4) were noticed in response to acid spraying. Gibberellic acid treatment improved leaf relative water content. The reverse held true for plants sprayed with distilled water. Single factors as well as their interaction significantly affected leaf RWC as indicated by F value (Table 2). The role of pH was dominant and that of GA3 was subdominant.

DISCUSSION Exposure of sunflower shoot to an acid mist of pH 2.0 reduced the stability of leaf membranes.

Leaf discs taken from acid sprayed plants were more leaky (high percentage of injury) under dehydration and heat stress than those sprayed with distilled water. Such effect could be due to destroy of cell membrane integrity and increase the sensitivity of leaf membranes to dehydration (40% PEG) and heat 51 ºC stress due changes in leaf membrane bilayer under influence of acid pH [31 ]. If cell membrane integrity is damaged, outward leakage of symplastic ion may increase concentration of apoplastic ions and enhancing pattern of increasing ion leakage [32].

The acid spray in liquid form produced droplets too large to enter through stomata. The first step in damage was an effect on surface wax [33]. Changes in the surface waxes due to acid rain are accompanied not only structural damage [34] but by quantitative and qualitative changes in the secondary substances embedded therein. Therefore potential damage caused by acid spray may be signaled by physical indicators which monitor the progress of damage from leaf surface inward.

Evidence exists for threshold pH surface moisture, in the range 2.5 to 4.0 for deciduous broad leaf and wheat plants, below which visible leaf damage in the form of lesions becomes apparent [9, 35]. In our study no visible injury to the surface of the sunflower foliage occurred during the misting treatment of pH 2.0. However, as water droplets dried after the cessation of misting small pale-green lesion appeared on the leaves of plants receiving this treatment.

In the present study spraying with HCl and H 2SO 4 solutions significantly decreased leaf chlorophyll content and dry mass production which is in

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Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants accordance to previous findings [4, 7, 9]. This can be proposed to be a consequence of the oxidative damage to important photosynthetic cells [36] which contain an array of photosensitizing pigments that produce and consume oxygen [37]. Cao et al., [1 0] reported that weak acidity (pH 4.0-5.0) simulated acid rain had less effect on the plant biomass, leaf chlorophyll content, photosynthetic characteristic and yield. However, Porter and Sheriden [38] found that rain with a pH 2.0 to 6.0 did not affect chlorophyll content in legumes until visible damage was present. Contrarily, sprayed sunflower plants with HNO 3 solution had much more chlorophyll than the plants sprayed with distilled water, probably due to enhancement of chlorophyll formation by NO 3- ion.

Soluble sugars of sunflower plants sprayed with HCl and H 2SO 4 solutions were substantially lower than unsprayed plants. Reduction in soluble sugar content in acid misted plants probably due to effect of acid solution on photosynthic activity through either reduction in CO 2 fixation and photosynthetically active leaf area and suppression the synthesis of sugars [9,1 0] or through chlorophyll reduction (Fig. 2) and stimulation of H 2O 2 accumulation. H 2O 2 acts as a local or systemic signal for leaf stomata closure [39].

On the other hand, plants sprayed with NHO 3 solution had higher contents of soluble sugars than unacidified plants. This could be due to higher chlorophyll content (Fig. 1 ) which helped to keep photosynthesis at a higher rate with an accumulation of metabolic products.

Soluble proteins were lower in plants received acid solution than in control plants (sprayed with distilled water). Plants receiving HNO 3 and HCl solution had more free amino acids compared to control. Such an effect could be due to inhibition of amino acid incorporation in protein synthesis under acidic conditions. On the contrary, Mai and other (2008) found that soluble protein and total free amino acids contents in wheat leaves decreased with increasing acidity of acid rain.

The ability of plant to overcome the effect of the acid rain stress and to sustain its productivity may be related to scavenging of stress- induced toxic oxygen radical such as H 2O 2 (hydrogen peroxide), H â&#x20AC;˘ (hydroxyl radical, etc). Spraying with HNO 3 and H 2SO 4 solutions enhancing accumulation of H 2O 2 and lipid peroxidation could be due to decreased anti-oxidation enzymes activities and non enzyme antioxidant contents under acid rain stress [1 0]. As

Foyer and other [40] stated, H 2O 2 is a strong oxidant that initiated localized oxidative damage leading to disruption of metabolic function and losses of cellular integrity at sites where it accumulates.

The ability of plants to evolve mechanisms to detoxify toxic chemicals produced inside the cytoplasm allows them to grow with success in adverse environmental conditions [41 ]. Air pollution stress may decrease or increase the concentration of ascorbic acid in plant leaves. [42]. Plants more tolerant to air pollution seem to contain more ascorbic acid when compared with less tolerant plants [43].

Our results indicated that sunflower plants accumulated less ascorbic acid in response to acid spraying. This means that sunflower plants are less tolerant to air pollution.

Phytohormones are active members of the signal compounds involved in the induction of plant stress responses [44]. They not only regulate plant growth and development, but also increase plant resistance to various environmental stress conditions [45]. In this paper, gibberellic acid appears to give sunflower plants some protection against acid solution of pH 2.0. Spraying with GA3 protected the plant membranes from dehydration and heat stress injury where leaf discs excised from GA3 treated plants were more stable (less injured) than the untreated plants. This protection conferred by AG 3 is presumably due to its role in protecting the plant lipid from peroxidation through decreasing malonyldialdehyde (MDA) content (Fig. 3).

Contrarily to acid mist, treatment with GA3 increased ascorbic acid contents in plants received HNO 3 and H 2SO 4 acid solution. Ascorbic acid is widely utilized in cell metabolism and is needed to synthesis hydroxyl proline-containing protein [46], to achieve cell division and cell expansion [47]. The metabolite of ascorbic acid (Asc A) may directly affect the synthesis of thymidine and uridine nucleotides, both of which are needed for nucleic acid synthesis [48].

Acidified plants receiving GA3 had more chlorophyll content concurrent with marked increase in soluble sugars and dry mass content. Enhancement of chlorophyll content by spraying with GA3 has been recently reported [49]. This may well be attributed to the GA3-generated enhancement of ultra structural morphogenesis of plastids coupled with retention of chlorophyll and delay of senescence caused by the hormone treatment [50]. The action of GA3 promotes sucrose synthesis within the leaf

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Sayed & Gadallah Effect of gibberellic acid on acid misted sunflower plants through their positive effect on fructose-1 ,6-biphosphatase and sucrose phosphate synthase [51 ].

According to Kozlowska et al. [52] GA3 may stimulate the photosynthetic activity from the beginning of shoot growth and are thus responsible for enhancement of yield. Khan et al. [53] have shown that GA3 spraying on mustard plants contributed 33.5% more leaf area and eventually led plants to have a better chance of trapping sunlight and producing more dry matter. The 33.5% increase in leaf area contributed to 27.1 % increase in dry matter production.

GA3 application increased sunflower tolerance to acid mist through stimulation of amino cid accumulation (Fig. 2) which has been reported to play important role (especially proline) in regulating the cytosolic acidity [54]. Generally, misted sunflower plants treated with GA3 had higher soluble proteins than the untreated plants. GA3 is known to have a secondary enhancement effect on protein content through the intensification of nitrate reductase activity [55]. Stimulation of the enzyme protein synthesis by GA3 stimulates the overall protein synthesis [56].

GA3 action in alleviating oxidative stress generated due to the production of reactive oxygen species under acid mist conditions is well documented in this paper where treatment with GA3 prevented the enhancement of H 2O 2 and lipid peroxidation caused by acid mist.

In conclusion, the results of this study clearly indicate that single acid mist treatment induced oxidative stress related to membrane damage.

GA3 application provides an attractive approach to cope acid mist. It improves sunflower tolerance by reducing reactive oxygen (H 2O 2), increasing ascorbic acid content and maintaining enzyme activities. It also counteracts with acid mist by improving membrane stability and water status in leaves, which ultimately leads to better growth.

It is therefore, possible that foliar GA3 could be useful tool in promoting growth under acid rain condition.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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Nasri N, Mahmoudi H, Baatour O, M’rah S, Kaddour R, Lachâal M. Effect of exogenous gibberellic acid on germination, seedling growth and phosphatase activities in Lettuce under salt stress. Afr J Biotechnol. 201 2; 11 : 11 967-11 97.

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Premabatidevi RK. Effect of IAA, GA3, and kinetin on nitrate reductase and nitrite reductase in the leaves of a tree legume. Indian J Plant Physiol. 1 998; 3: 971 01 .

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Journal of Biology and Earth Sciences

MEDICINE

REVIEW

Molecular basis of human obesity - literature review AgataTomaszewska1 ,2, Przemysław Kopczyński 1 , Rafał Flieger1 , Anna Thielemann 3 1The

Centre for Orthodontic Mini­implants of the Chair and Clinic of Maxillofacial Orthopaedics and Orthodontics, Poznań University of Medical Sciences, Poland

2The

Chair and Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, Poland 3The

Chair and Department of Laboratory Diagnostics, Poznań University of Medical Sciences, Poland

ABSTRACT

Obesity has become a serious health problem as well as economic and social challenge in the highly developed societies. In 201 0 there were over 1 billion overweight people worldwide and ca. 475 million were classified as obese with the BMI ≥ 30. That global health problem became an alarming issue affecting developed and developing countries alike. Genetic factors are attributed a special role in the etiopathogenesis of obesity. Recent data suggest that mutations and polymorphism identified in various genes contribute to a higher predisposition to obesity. In this review, we focus on the human mono- and polygenic obesity syndromes. The purpose of this work is to present, based on the literature review, the most recent findings concerning genetic causes of obesity.

Key words: Body Mass Index; Human obesity; Genetic markers; Mutation; Polymorphism.

J Biol Earth Sci 201 3; 3(2): M48-M51

Corresponding author:

Agata Tomaszewska Poznań University of Medical Sciences, The Centre for Orthodontic Mini-implants of the Chair and Clinic of Maxillofacial Orthopaedics and Orthodontics Bukowska 70, str., 60-81 2 Poznań, Poland e-mail: agatatomaszewska@ump.edu.pl Original Submission: 22 October 201 3; Revised Submission: 06 November 201 3; Accepted: 07 November 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Tomaszewska et al. Molecular basis of human obesity - literature review

INTRODUCTION

REVIEW

Obesity has become a serious health problem as well as economic and social challenge in the highly developed societies. In 1 997, the World Health Organisation (WHO) officially declared obesity to be a global epidemic. The report of the International Obesity Task Force (IOTF) estimated that in 201 0, there were over 1 billion overweight people worldwide and ca. 475 million were classified as obese with the BMI â&#x2030;Ľ 30 [1 ]. That global health problem became an alarming issue affecting developed and developing countries alike. An upward trend was recorded almost in all of the age groups, irrespective of the gender and ethnic origin. Nevertheless, the results of the analysis concerning populations of the USA and the Western European countries are especially worthy of note as they are characterised by the highest body mass indices (BMI) [2].

The research conducted in the recent years indicated that in the Polish population ca. 67% of persons have been found to be overweight whereas obesity has been identified in ca. 30% of women and 20% of men. A noticeable increase in obesity among children, parallel to obesity among adults, is another worrying issue, recorded especially in the last two decades. The IOTF report from 201 0 states that as many as 200 million children at the school age have excessively high weight, 40-50 million of which have been classified as obese [3, 4]. A considerable intensification of the obesity phenomenon is related to various environmental factors, minimalized physical activity and consumption of high-calorie foods dictated by wrong eating habits.

As regards the direct physiological cause of obesity, energy oversupply and reduced expenditure are deemed to be the main factors [5].

Quick development of the genetic research in the last two decades allowed to define the genetic causes of numerous diseases. At present, a lot of attention is given to studies on hereditary causes of increasingly more common problem of obesity.

Genetic factors are attributed a special role in etiopathogenesis of obesity. Mutations identified in various genes contribute to a higher predisposition to excessive accumulation of adipose tissue [6].

The hypothesis of the â&#x20AC;&#x153;thrifty genotypeâ&#x20AC;? states that the tendency in man to accumulate more adipose tissue was preferred in evolutionary terms, as it allowed our ancestors to survive through the periods of famine. The above hypothesis may be confirmed by the results of numerous population research of e.g. inhabitants of small islands on the Pacific Ocean, the Pima Indians or the societies of the Kingdoms of Tonga and Samoa. Those populations are characterised by a very high obesity index and extremely effective adaptive mechanisms for quick storing of energy excesses in the form of triglycerides deposited in the adipose tissue [7].

A considerable importance of the genetic mechanisms was also confirmed by many experimental studies regarding etiology and pathogenesis of obesity, conducted on model laboratory animals (among others, the gene knockout mouse) [8].

The effects of oversupply of the physiological energy requirements are detrimental to health and disrupt the energy homeostasis in the system.

Obesity has enormous impact on the glucose homeostasis and lipid homeostasis in the organism.

Currently, it is known that the obesity effects increase the risk of co-occurrence of numerous pathologies, both those which develop quickly in relation to obesity, as well as those which are deferred in time and may appear even after many years. Given that fact, obesity was related with incidence of numerous diseases, e.g. dyslipidemia, arterial hypertension, insulin resistance and hyperglycemia. It also contributes to a higher incidence rate of diabetes, atherosclerosis and cardiovascular diseases [9].

The results which proved the discovery in model laboratory animals of many various mutations conditioning increased predisposition to develop obesity and other metabolic disorders being a direct consequence of a malfunctioning body energy expenditure, aroused a great interest among the

MATERIAL AND METHODS The purpose of this work is to present, based on the literature review, the most recent findings concerning genetic causes of obesity. The analysis covered 60 publications from the library database from the last 1 0 years. The search criterion were the following phrases: genetic markers, mutations, human obesity, polymorphism. Finally, 20 of the 60 publications were selected for analysis.

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Tomaszewska et al. Molecular basis of human obesity - literature review scientists worldwide. Obesity, along with the voracity effect were observed in model animals with induced genetic changes in the leptin/melanocortin pathway [1 0]. In the case of the Mc4r knockout mouse, the scientist identified an intensified process of depositing adipose tissue and increase in the amount of food intake, leading to obesity in such animals. Contrary to the effects observed in the Mc4r knockout mouse, the mice with Mc3r knockout were characterised by a unique phenotype, i.e. higher percentage of adipose tissue whereas the body weight remained unchanged. Moreover, they were also marked by decreased physical activity and hyperleptinemia. On that account, many scientists directed their effort to search for new as well as analogous polymorphisms and mutations in man.

Such research focuses primarily on genes and their protein products participating in various metabolic pathways, mainly related to maintaining the correct level of the system’s energy management.

Individual cases of monogenic obesity have been explained with identified rare causative mutations occurring in various genes, e.g. in the gene of leptin (LEP), leptin receptor (LEPR), proopiomelanocortin (POMC) and melanocortin 4 receptor ( Mc4r). Unlike the unambiguous findings related to Mc4r, the role of the melanocortin 3 receptor ( Mc3r) in the body weight regulation is still being discussed [11 , 1 2].

Monogenic obesity belongs to a group of relatively rare yet significant diseases, as it provides an opportunity to thoroughly analyse the mechanisms responsible for appetite control and regulation.

Monogenic obesity is inherited in line with the Mendel’s laws. As a result of experiments conducted on mice with hereditary significant obesity (homozygotes ob/ob, db/db and fat – with alleles encoding leptin, leptin receptor and carboxypeptidase respectively), it was possible to find analogous mutations in man [1 3].

Food intake and body weight are controlled by the centres located in various regions of the central nervous system (CNS). The arcuate nucleus of hypothalamus, responsible for that mechanism, is conditioned by a number of factors, including an important role of the melanocortin receptors (MCR).

Malfunctioning of such mechanisms may result in serious food intake disorders which may lead to obesity. As it has been indicated so far, neural melanocortin 3 and 4 receptors affect various aspects of maintaining the energy homeostasis in

the organism, and neuroendocrine disorders, driven by melanocortin receptors mutations, have an obesogenic effect [1 4, 1 5].

To date, the scientists have described a number of mutations of various genes leading to monogenic obesity, e.g. mutations in the genes of leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor ( Mc4r), proopiomelanocortin (POMC), prohormone convertase 1 (PCSK1), neurotrophine 2 receptor (NTRK2) and brain-derived neurotrophic factor (BDNF) [1 6].

The mutations in the genes participating in hypothalamic regulation of appetite, i.e. LEP, LEPR and Mc4r, are of the highest importance. As a result of incorrect leptin functioning, driven both by lack of that gene expression and defect of its receptor, hyperphagia occurs and obesity ensues in the first months of life. It has been estimated that obesity related to the defect of melanocortin 4 receptor is the most common form of monogenic obesity, Mc4r mutations are responsible for 2.4%-4% of significant obesity (BMI ≥ 35). Other mutations concern the proteins of the melanocortin pathway, participating in the neurogenesis and correct functioning of the hypothalamic neural network [1 7].

As it has been demonstrated, clinical observation indicating more frequent cases of family obesity inspired to search for the genes correlated with that trait. Nowadays it is known that a correct body weight has largely a polygenic basis [1 8]. This means that obesity may be a result of a number of genetic mutations, of which every mutation analysed separately indicates an insignificant effect on increase in the body weight. Currently, the number of genes and genetic markers has exceeded 1 00, and the identified chromosomal regions associated with obesity are located on all the chromosomes except for the chromosome Y. In addition, heritability (h 2) of obesity is estimated between 0.4 and 0.7. To date, a vast number of regions associated with polygenic obesity have been identified with the number of candidate genes still growing. This applies first of all to mutations of genes whose products are responsible for maintaining the correct energy homeostasis in the organism, affect the hypothalamic centres regulating the appetite and are related to the correct fat thermogenesis and metabolism. Among the genes in this group there are: FTO, INSIG2, PPARγ, SLC6A14. What is interesting, hereditary factors have also an important

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Tomaszewska et al. Molecular basis of human obesity - literature review role in forming psychophysical behaviours. Personal responses and increased susceptibility of some individuals to environmental stimuli conducive to gaining weight are also conditioned genetically [1 9, 20].

CONCLUSIONS To sum up, most frequently human obesity is the result of genetically conditioned pathophysiological, psycho-emotional and behavioural responses leading to excessive of accumulation of adipose tissue in the organism. It should be also emphasised that the causes of obesity are very complex and various genes of susceptibility to obesity need to be affected also by external environmental factors to result in the disease expression.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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1 3. Pillot B, Duraffourd C, Begeot M, Joly A, Luquet S, Houberdon I, et al. Role of hypothalamic melanocortin system in adaptation of food intake to food protein increase in mice. PLoS ONE. 2011 ; 6(4): e1 91 07.

1 4. Tao YX. Mutations in melanocortin-4 receptor and human obesity. Prog Mol Biol Transl Sci. 2009; 88: 1 73-204.

1 5. Tao YX. Mutations in the melanocortin-3 receptor (MC3R) gene: Impact on human obesity or adiposity.

Curr Opin Investigat Drugs. 201 0; 11 (1 0): 1 092-1 096.

1 6. Choquet H, Meyre D. Molecular basis of obesity: current status and future prospects. Curr Genomics.

2011 ; 1 2(3): 1 54-68.

1 7. Męczekalski B, Czyżyk A, Warenik-Szymankiewicz A. Rola genów w powstawaniu otyłości. Współczesne poglądy, patogeneza, aspekty kliniczne. Endokrynol Otyłość Zaburz Przemiany Materii. 2008; 5(1 ): 27-37.

1 8. Choquet H, Meyre D. Genomic insights into earlyonset obesity. Genome Med. 201 0; 2(6): 36.

1 9. Clee SM. Genetic gains on the obesity and metabolic disease fronts. Clin Genet. 2009; 76(3): 236-241 .

20. Braud S, Ciufolini M, Harosh I. ‘Energy expenditure genes' or 'energy absorption genes': a new target for the treatment of obesity and Type II diabetes. Future Med Chem. 201 0; 2(1 2): 1 777-1 783.

1 . http://www.iaso.org/iotf/ 2. Mutch DM, Clement K. Unraveling the genetics of human obesity. PLoS Genet. 2006; 2(1 2): e1 88.

3. Segal ME, Sankar P, Reed DR. Research issues in genetic testing of adolescents for obesity. Nutr Rev. 2004; 62(8): 307-320.

4. Fichna P, Skowronska B. [Obesity related complications in children and adolescents]. Endokrynol Diabetol Chor Przemiany Materii Wieku Rozw. 2006; 1 2(3): 223-228.

5. Kociniak T, Rosołowski M, Dziemianowicz M, Łukaszyk A. Otyłość – epidemia XXI wieku? Gastroenterologia Polska. 2005; 1 2(2): 1 49-1 53.

6. Farooqi S, O'Rahilly S. Genetics of obesity in humans. Endocr Rev. 2006; 27(7): 71 0-71 8.

7. Konarzewski M. Ewolucja otyłości. Nauka. 2006; 4: 85-96.

8. Lutz TA, Woods SC. Overview of animal models of obesity. Curr Protoc Pharmacol. 201 2; 5,Unit 5: 61 .

9. Clement K. Genetics of human obesity. C R Biol.

2006; 329(8): 608-622.

1 0. Speakman J, Hambly C, Mitchell S, Król E. Animal models of obesity. Obes Rev. 2007; 8 Suppl 1 : 55-61 .

11 . Farooqi IS. Monogenic human obesity. Front Horm Res. 2008; 36: 1 -11 .

1 2. O’Rahilly S, Farooqi IS, Yeo GSH, Challis BG. Minireview: human obesity - lessons from monogenic disorders. Endocrinol. 2003; 1 44(9): 3757-3764.

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Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India Poonam Bhadja, Ashokkumar Vaghela Department of Zoology, Kotak Institute of Science, Saurashtra University, Gujarat, India

ABSTRACT

Water quality has become a major concern due to ever increasing human developmental activities that over exploit and pollute the water resources. The physico-chemical parameters like pH, EC, DO, BOD, alkalinity, hardness, calcium and magnesium were analyzed during monsoon and post-monsoon season. A study on physical, chemical and biological characters of River water and its suitability for drinking purpose was carried out of reservoir water of Rajkot, Gujarat. The results of the present study have been discussed it is clear that the water is not highly polluted, but the variations in physico-chemical parameters were observed as seasonally. The recorded range of physico-chemical parameters were within the maximum permissible limit.

Key words: Physico-chemical parameters; Reservoir; Seasonal variations.

J Biol Earth Sci 201 3; 3(2): E1 2-E1 7

Corresponding author:

Ashokkumar Vaghela Department of Zoology, Kotak Institute of Science, Saurashtra University, Rajkot-360005, Gujarat, India.

Phone: +91 9898242339 E-Mail: ashokvaghela2@gmail.com Original Submission: 1 5 June 201 3; Revised Submission: 31 July 201 3; Accepted: 01 August 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, E1 2-E1 7

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Bhadja and Vaghela Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India

INTRODUCTION Water is the base of life and development. The wetland forms unique biological fresh water ecosystem on the planet earth. These water bodies store the freshwater from adjoining are during rainy season. It plays an important role in any ecosystem, hydrology of area and economy. They provide the habitats for migratory birds, aquaculture, plants, animals and microbes. Water is one of the major components of environmental resources [1 ].

Freshwater is a natural home of innumerable living things, many of them harmless or even beneficial, some of them directly or indirectly injurious to man. The environmental pollution affects the general quality of our health [2]. Several studies have been made on the limnology of freshwater bodies in India [3-5]. Water resources in India have reached a point of crisis due to unplanned urbanization and industrialization [6]. Urbanization has directly negative impacts on water bodies.

Therefore now a days freshwater has become a scare commodity due to over exploitation and pollution [5]. River water is one of the most important and widely distributed natural resources which are considered as supplemental resource to meet the domestic, agriculture and industrial requirements. The present work is aimed in assessing the reservoir water quality with respect to drinking purpose.

Gujarat receives throughout the year is about 276 mm.

The study sites, Aji Reservoir (22º1 5'34'' N, 70º50'56'' E), Nyari reservoir (22º21 '45'' N, 70º40'1 5'' E) and Lalpari Lake (22º1 8'11 '' N, 70º51 '7'' E) at Rajkot City in Gujarat was selected for present investigation (Figure 1 ). The lake is saturated with fish, many of whom are overgrown due to fishing restrictions. These sampling locations are one of the most exotic natural spots on the outskirts of the city.

Collection of water

The present study deals with few physical and chemical parameters of the water to check the present status of water quality of sampling site. The study was conducted during January 2011 to December 201 2. For water sample collection plastic sample bottles having capacity of one liter were

MATERIALS AND METHODS Study area

The present communication deals with the freshwater reservoir around Rajkot city. Rajkot is situated in the middle of the peninsular Saurashtra in central plains of Gujarat State of Western India.

It lies between latitude 22º1 8’ N and longitude 70º51 ’ E. It has an area of 1 04.86 sq. kms. Rajkot is an important urban centre, centrally located in Saurashtra region, in the State of Gujarat. The Rajkot city experiences a semi-arid climate that has three seasons viz. summer, winter and monsoon.

Summers are very hot and dry and the monsoon is accompanied by moderate rainfall. The weather condition of the city is that summer temperature ranges between 32-44ºC and winter temperate ranges between 1 8-25ºC. The rainy season stretches from the month of July to the month of September. The average rainfall that this place of

Map of the study locations along Rajkot City, Gujarat, India.

Fig. 1.

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Bhadja and Vaghela Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India filled without disturbing the substratum to avoid the loose sediments in sample. Samples were collected from surface (1 -2 cm). After collection of samples, these bottles were labeled and possible efforts were made to transport them to the laboratory as earlier as possible. The samples for DO and BOD analysis were collected from surface from the Sampling site in separate BOD bottles. Two such bottles were used for each sample. One was fixed on the spot immediately after the collection following Winkler method [7], and the second bottle containing water was kept in darkness at 4ºC (in iceboxes) till it reached the laboratory.

Water analysis methods

Physical and chemical analysis of the samples was done according to Standard Methods as per APHA, [8, 9] and Trivedi and Goel [7]. The values obtained were compared with standards prescribed

by WHO [1 0] and BIS [11 ]. Few parameters such as temperature, pH and Electric Conductivity were recorded on the site by their respective probes.

Chemical parameters such as Dissolved Oxygen (DO), Biological Oxygen Demand (BOD), Total Solid, Total Dissolved Solids (TDS), Total Suspended Solids (TSS), Hardness, Calcium, Magnesium and Free CO 2 were then dealt in the laboratory. The following table reveals the parameters, their units and the methods used for their analysis.

Table 1.

RESULTS AND DISCUSSION The results of physic-chemical analysis of three freshwater reservoirs for different seasons are given in Tables 1 , 2, and 3. The data presented are discussed on the basis of three seasons.

The temperature of water varied between 21 .6ºC and 23.7ºC at the sampling site Aji Reservoir. At the sampling site Nyari it ranged between 21 .65ºC and 23.55ºC and at sampling site Lalpari ranged between 21 .85ºC and 23.80ºC. In all the three sampling locations high temperature was recorded during summer season and lower temperature recorded during winter season, which is a normal feature in freshwater reservoirs. The water temperature is one of the most important physical characteristics of aquatic ecosystem, as it affects the organisms [5]. It affects a number of water quality parameters that is one of the concerns for domestic, environmental, industrial and agricultural applications [1 2]. The lowest pH values were recorded during monsoon season, which implies the influence of run-off water entering into the water bodies. The desirable limit of pH recommended by drinking water specification Indian Standard – IS 1 0500: 1 991 is 6.5–8.5 [11 ]. The average pH value at Aji Reservoir was 8.1 0 whereas at Nyari and Lalpari reservoir it was 8.1 4 and 8.22 respectively.

The total solids at sampling site Aji reservoir ranged between 476 and 564.5 mg/l and the

Physico-chemical parameters of Aji Reservoir.

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Bhadja and Vaghela Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India Table 2.

Physico-chemical parameters of Nyari Reservoir.

Table 3.

Physico-chemical parameters of Lalpari Reservoir.

values at Nyari reservoir ranged between 473 and 643 mg/l, while at the sampling site Lalpari reservoir it ranged from 607 to 687 mg/l. Maximum values of total solids were recorded during monsoon season at all the sampling locations. Run-off water, which carries dissolved solids and also organic wastes from garbage dumping, contributes to higher total solids [1 3]. The total dissolved solids at the Aji reservoir varied between 439 and 535.5 mg/l and at

Nyari reservoir ranged between 444.5 and 61 4 mg/l.

At the sampling site Lalpari reservoir it was ranged between 51 6 to 607 mg/l. Maximum values of total dissolved solids were recorded during monsoon season at all the sampling sites. The desirable level of total dissolved solids is 500 mg/l, presence of excess total dissolved solids may cause gastrointestinal irritation when consumed [1 3]. It elevates the density of water and reduces solubility

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Bhadja and Vaghela Hydrobiological studies on freshwater reservoir of Saurashtra, Gujarat, India of oxygen that may prove lethal to aquatic life [5].

The total suspended solids were ranged between 29 and 30 mg/l at sampling site Aji reservoir, at the Nyari reservoir it was ranged between 25 and 32 mg/l. It was ranged between 59 and 91 mg/l at the sampling site Lalpari reservoir (Table 3). The concentration was high during monsoon season, which may be due to addition of solids from runoff water to the reservoir [5].

The electrical conductivity of the water samples ranged between 584 and 832.5 ÂľS/cm throughout the study period at all the sampling sites. Water of higher conductivity may be used with suitable amendments and precautions, but under normal conditions they are harmful to the soil structure and their continuous use will result in salinity hazard, with ultimate effect on plant growth [1 4]. There is currently no official guideline as to what is considered safe level for conductivity [1 5]. However, the conductivity of most freshwaters ranged from 1 0 to 1 000 S/cm, but many exceed 1 000 S/cm. especially in polluted waters, or those receiving large quantities of land run-off [5, 1 6].

The dissolved oxygen is an important aquatic environmental factor, which influences the health of an aquatic ecosystem. The higher value of dissolved oxygen may be due to the influence of run-off water from monsoon rain [1 3]. The recorded dissolved oxygen range was within the maximum permissible limit [1 7]. It was found that higher dissolved oxygen values were observed in monsoon may be due to higher water temperature, higher biological oxygen demand on account of decomposition of organic detritus during this period.

It revealed that the quality of water at the residential areas was found to be safe and could be used for domestic purpose and without any treatment [1 8].

Biological oxygen demand is the measure of quantity of oxygen required by bacteria and other microorganisms under aerobic condition in order to biochemically degrade and transform organic matter present in the water bodies [5].

The total hardness values at Aji reservoir ranged between 221 and 258 mg/l, whereas at Nyari reservoir it was 231 .5 and 251 mg/l and at the sampling site Lalapari it was 243.5 and 265 mg/l.

Total hardness of water is due to the concentration of salts. In particular, it is due to the concentration of multivalent metallic ions of calcium and magnesium.

The desirable limit of total hardness is 300 mg/l.

Hardness has no known adverse effects on health;

however some evidences have been given to indicate its role in heart diseases [1 9]. Calcium and magnesium are common constituents of natural water and important contributor to the hardness of water. The calcium concentration in water samples ranged from 45.0 mg/l to 62.4 mg/l. and in case of magnesium, it was ranged between 35.63 mg/l and 42.72 mg/l at Nyari reservoir. The results revealed that various physico-chemical variables were well within acceptable limits of water quality [20]. Perona et al. [21 ] suggested that if physico-chemical variables did not show wide range of variation which is due to the closely associated with the lithological composition of the river basin. The physical and chemical characteristics of water showed seasonal fluctuations interacting with one another and have a combined effect on animals and plants [22]. Factors controlling the composition of natural waters are extremely varied and include physical, chemical and biological processes [23].

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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2007; 11 (2): 91 -93.

1 3. Chennakrishanan C, Stephen A, Manju T, Raveen R. Water quality status of three vulnerable freshwater lakes of Suburban Chennai, India. Ind J Environ Ecoplan. 2008; 1 5(3): 591 -596.

1 4. Dutta S, Chowhan P. Assessment of groundwater quality of Masuda tehsil, Ajmer district, Rrajasthan.

Ecoscan. 2009; 3 (3&4): 281 -284.

1 5. Karikari AY, Asante KA, Biney CA. 2007. Water quality characteristics at the estuary of Korle Lagoon in Ghana. Unpublished paper. CSIR-Water Research Institute, P.O. Box M32, Accra-Ghana.

1 6. Chapman D. 1 992. Water quality assessment: a guide of the use of biota, sediments and water in environmental monitoring. University Press, Cambridge, pp: 585.

1 7. WHO. 1 984. Guidelines for water quality. Vol. 1 .

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1 8. Sathya R, Shankar P. Status of lake water quality in Karavetti. J Basic Appl Biol. 2009; 3(1 &2): 36-41 .

1 9. Peter AK. 1 974. Sources and classification of water pollutants in industrial pollution. Edlrving Sax. Van Nostrand Reinhold Company.

20. Boyd CE, Tucker CS. 1 998. Pond aquaculture water quality management. Kluwer Academic Publishers, London.

21 . Perona E, Bonilla I, Mateo P. Spatial and temporal changes in water quality in a Spanish river. Sci Total Env. 1 999; 241 : 75-90.

22. Odum EP. 1 971 . Fundamentals of ecology. 3rd ed.

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23. Boyd CE. 1 981 . Water quality in warm water fish ponds. Craftmaster Printer, Inc. Opelika, Alabama.

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TMKARPIŃSKI

ISSN: 2084-3577

PUBLISHER

EARTH SCIENCES

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Plutonites of Wadi Um Arka, Allaqi region, South Eastern Desert, Egypt: remote sensing and geochemical aspects Ezzat Abdel Rahman 1 , Nedal Qaaud 2, Ashraf Emam 1 , Nagdi M. Abdou 3 1Geology 2Geology

Department, Faculty of Science, Aswan University, Egypt Department, Faculty of Science, Al­Azhar University, Gaza, Palestine 3Nuclear Materials Authority, Maadi, Cairo, Egypt

ABSTRACT

The late Proterozoic basement rocks of island-arc assemblage and syn, late to post-tectonic intrusions occupy Wadi Um Arka area. Remote sensing data analysis has been applied using band ratio and fusion techniques to discriminate the studied plutonites from their country rocks and to produce more detailed (1 :20,000) image map for these rocks. The syn-tectonic granitoids are mainly granodiorite in composition, whereas the late to post tectonic intrusions are differentiated into felsic granitoid and mafic gabbroic rocks. Microscopically, biotite granites and muscovite leucogranites represent the late to post-tectonic granitoids, while the younger gabbros are distinguished into olivine gabbro, pyroxene gabbro and anorthosite. The syn-tectonic granitoids show calc-alkaline, peraluminous and sodic affinity.

They have implaced during pre-plate collision stage in continental arc regime under high pressure (> 5 k-bars), compressional stresses and high temperature (800-840ºC) at greater depths more than 30 km. The biotite granites show calc-alkaline to alkaline, metaluminous to slightly peraluminous and potassic affinity. They have emplaced during late-orogenic regime in compressional suite. The muscovite leucogranites have alkaline, peraluminous to slightly metaluminous and potassic affinity. They have emplaced during syn-collision stage. These younger granitoids were formed in compressional suite under relatively low to moderate pressure (1 -5 k-bars) and temperature (760-800ºC) conditions and at moderate depths (20-30 km). The younger gabbros of Wadi Um Arka area were derived from subalkaline basic magma exhibiting tholeiitic nature with mild calc-alkaline tendency. They have emplaced in compressional subduction-related environment having characters of the island-arc and active continental margin.

Key words: Plutonites; Remote sensing; Geochemistry; Granites; Gabbros; Um Arka; Allaqi.

J Biol Earth Sci 201 3; 3(2): E1 8-E38 Corresponding author:

Ezzat Abdel Rahman Geology Department, Faculty of Science, Aswan University, Egypt E-mail: amezzat201 0@gmail.com Original Submission: 05 June 201 3; Revised Submission: 1 8 July 201 3; Accepted: 29 July 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, E1 8-E38

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

INTRODUCTION Wadi Allaqi region is described as a foreland basin in which a thick pile of molasse sediments accumulated above shelf sediments containing limestone layers [1 , 2], a major fault-zone [3], a suture-zone between Aswan and Gabgaba terranes [4], an ophiolitic mélange [5]. Wadi Allaqi region comprises a well foliated, northwest trending belt of metasediments and metavolcanics preserving thrust duplex structures, interwoven with ophiolitic nappes and intruded by syn-, late-, and post-tectonic gabbro-granite complexes [6-9]. The area is covered by ultramafic rocks and highly sheared derivatives, amphiboliltes, metasediments, metavolcanics and metatuffs. These rocks are intruded by foliated gabbro-diorites, syn- to late-tectonic granitoids and gabbros [1 0]. The granitoid rocks of Wadi Heisurbah-Wadi Quleib area, northwest of Wadi Allaqi district are subdivided to granodiorites, monzogranites and alkali granites [11 ].

Um Arka (Hariari) gold mine area is located within the northwestern part of Wadi Allaqi region between latitudes 22º54'00'' & 22º58'30'' N and longitudes 33º25'00'' & 33º29'30'' E (Fig. 1 ).

Precambrian basement rock units of island-arc assemblage and syn-, late- to post-tectonic

intrusions cover the area. Two main shear zones, trending NW-SE and ENE-WSW, dissect the area.

Also, several faults, extending NW-SE, ENE-WSW and NE-SW traverse the rocks covering this area.

Moreover within the shear zones, the rocks are well foliated with foliation planes trending mainly in the NW-SE and ENE-WSW directions.

The present paper aims at studying geologic setting of Wadi Um Arka area by remapping and construction of more detailed geologic map at scale 1 : 20,000 using band ratio and fusion techniques of remote sensing. Field investigation and petrography of each rock unit will be considered in detail to achieve this purpose. However, another target of this paper is studying the geochemistry of the granitoids and younger gabbros in the light of the geologic setting of the area to investigate their geochemical characters, the magma type from which they were derived and the tectonic environment within which they were formed.

RESULTS AND DISCUSSION Geologic setting

The late Proterozoic basement rocks occupying Wadi Um Arka area are classified and chronologically arranged as follows:

Island­arc assemblage rocks

Fig. 1.

Location map showing the studied area.

Outcrops of metasedimentary rocks and metavolcanics constituting the country rocks represent the island-arc assemblage rocks in the area under investigation. The metasedimentary rocks are fine-grained, cover about 1 2 km 2 in the southeastern part of the mapped area and form lowland outcrops as well as elongated sheets extending NW-SE parallel to the NW-trending shear zone. Their exposures are highly deformed (Fig. 2a) and exhibit well foliation planes having strike (from N30ºW to N60ºW) and dipping to the NE direction with dip angle (43º-83º). The metavolcanics and their metatuffs are covering about 5 km 2 within the

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a) Folded foliation in the metasediments, b) Elongated hills and moderately elevated masses of metavolcanics showing dome-like and conical shape, c) Subrounded mafic xenolith in the syn-tectonic granites, d) Sharp intrusive contact between the younger granite (YG) and gabbro intrusion (G), e) Microgranitic dykes invade the younger granites and form sharp crests with high elevation, f) Angular and subangular xenoliths enclosed within the microgranitic dykes.

Fig. 2.

study area and predominant in the northeastern corner. These rocks are represented mainly by metaandesites and andesitic metatuffs. They form small ridges, elongated hills and moderately elevated masses showing dome-like and conical shape (Fig. 2b). These metavolcanics are finegrained, massive rocks and occasionally dissected by various sets of joints leading to segmentation of the rocks.

Granites

The granitic rocks form the voluminous part of the mapped area covering about 50 km 2. They are predominant in the northern and northwestern parts, forming Gabal Um Arka. These granitoids are classified into two separated groups, namely as syn-tectonic (older) granitoids and late to posttectonic (younger) granitoids. The older granitoids are medium to coarse-grained and mainly granodiorite in composition, covering about 8 km 2.

They form irregular elongated masses as well as small isolated hills scattered in sandy plains throughout the southwestern and central sectors of the mapped area. They are dissected by several wadis and extensively weathered along joint planes forming low-lying hillocks and spherical masses containing mafic xenoliths with various sizes (Fig. 2c).

The younger granitoids range in composition from biotite granites of reddish and pink colours to

muscovite leucogranites of yellowish and whitish colours. These granitoids cover about 42 km 2 and form the main rock mass of Gabal Um Arka in the northern and northwestern parts of Wadi Um Arka area. Moreover, they form lensoidal intrusion which is discordant with the geological setting of the area in the southeastern part and extends westward further out the mapped area. In some places, their outcrops are cut by two or more sets of joints and extensively weathered forming cuboidal and spherical masses. Generally, the younger granitoids are medium to coarse-grained rocks showing intrusive contacts with the surrounding older granites and island-arc assemblage rocks.

Gabbros

The main intrusion of these rocks covers about 1 5 km 2 in the central and eastern parts of the mapped area and form lensoidal or oval mass invading the island-arc assemblage rocks and the granitoid rocks. They are massive, coarse-grained and characterized by sharp intrusive contact with the surrounding rocks (Fig. 2d). The ENE-trending shear zone is cutting across the gabbro intrusion at the contact with the Gabal Um Arka granitic rocks.

Within this shear zone, the gabbros are highly sheared, fractured and uralitized. Moreover, they were subjected to several hydrothermal alteration processes such as carbonatization, chloritization and ferrugination. Numerous ENE-trending quartz

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

a b

a) Lineament map of Hariari gold mine area showing the tectonic trends of different structural features affecting the area. b) A-F rose diagram shows the major tectonic trends (NW, NE & ENE) affecting Hariari gold mine area.

Fig. 3.

veins invaded the gabbro intrusion due to the open fissures and fractures filling with hydrothermal solutions cutting across this intrusion.

Several dykes and quartz veins invaded in the different rock units covering the study area, but they are concentrated in the northern, central and eastern parts along the shear zones. The dykes range in composition from basic to acidic, but the microgranitic dykes are the most voluminous and predominant variety. These microgranitic dykes have length ranging from 400 m to 3.8 km and thickness ranging from 200 m to 340 m. They are cutting across the metavolcanics, the gabbros, and the granitic rocks forming sharp crests with high elevations (Fig. 2e). They contain gabbroic xenoliths having various size (Fig. 2f) favoring that these dykes are post-date the emplacement of the granite and gabbro intrusions.

Lineament map for Hariari gold mine area has been traced from the aerial photographs and ETM+ landsat images. This map illustrates the tectonic trends of different structural features affecting the basement rock units occupying the study area (Fig. 3a). The Azimuth-Frequency rose diagram (Fig. 3b) illustrates three major tectonic trends.

These tectonic trends are the northwest (Red Sea or Gulf of Suez) trend, the northeast (Aualitic or Tibesti) trend and the east-northeast/westsouthwest (Syrian Arc) trend.

Petrography

The granodiorites have hypidiomorphic texture and consist mainly of plagioclase (An 28-35), quartz, hornblende, biotite and subordinate amounts of orthoclase (Fig. 4a). Sphene, apatite, zircon and iron oxides are the main accessory minerals (Fig. 4b) whereas epidote, chlorite, sericite and calcite are the secondary minerals. Microscopically, the biotite granites show hypidiomorphic as well as perthitic textures (Fig. 4c). They consist essentially of quartz, microcline, microcline perthite and plagioclase (An 1 5-1 7) with variable amount of biotite as the sole mafic mineral (Fig. 4d). Apatite, zircon, muscovite and iron oxides are the main accessories. The muscovite leucogranites are composed essentially of quartz, plagioclase (An 8-1 5), microcline and muscovite (Fig. 4e) whereas zircon, apatite and opaques occur as accessories. The potash feldspars show intergrowth with quartz giving rise to graphic texture (Fig. 4f).

The gabbroic rocks of Wadi Um Arka area range in composition from olivine gabbros, pyroxene gabbros to anorthosites. The olivine gabbros are coarse-grained rocks, composed essentially of olivine, calcic plagioclase (An 70-87) and pyroxene (enstatite and hypersthene) with subordinate amount of hornblende (Fig. 4g). Apatite and opaque minerals are the main accessories, whereas antigorite, chlorite and epidote represent the secondary minerals. Continuous and discontinuous

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a) Distorted lamellae in plagioclase and b) Prismatic zircon and apatite inclusions within plagioclase, in the older granite, c) Microcline perthite and d) Laths and flakes of biotite in the biotite granite, e) interstitial flakes and tiny crystals of muscovite and f) Micrographic texture in the muscovite granite, g) Coarse-grained olivine with diagnostic y-cracks, and h) Thin reaction rims surround the olivine crystals giving rise to corona structure in the olivine gabbros, i) Coarsegrained plagioclase partly to totally enclosed within pyroxene forming poikilitic textures and j) Uralitized pyroxene crystals in the pyroxene gabbros, k) Coarse-grained plagioclase and l) Plagioclase phenocryst exhibiting microfaulted twinning lamellae in the anorthosites, m) Fine to medium-grained quartz, plagioclase, orthoclase and scared biotite flakes in the microgranite dykes of Wadi Um Arka area.

Fig. 4.

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt thin rims surround the olivine crystals, especially at the borders of plagioclase and pyroxene, giving rise to corona structure (Fig. 4h).

The pyroxene gabbros are medium to coarsegrained rocks characterized by poikilitic textures (ophitic and subophitic) and composed essentially of plagioclase (An 59-68), pyroxene (hypersthene and augite) and actinolite enclosing pyroxene relics (Fig. 4i). Ilmenite, magnetite and sulphides are the accessories. Within the ENE-trending shear zone, the pyroxene gabbros are highly uralitized (Fig. 4j).

The anorthosites are coarse-grained, leucocratic and equigranular hypidiomorphic in texture (Fig. 4k). They are composed essentially of plagioclase (An 63-68) with subordinate amount of augite. Scarce amount of iron oxide grains disseminated throughout the rock as accessories.

Plagioclase euhedral crystals are internally cracked and the lamellae of twinning are twisted and microfaulted (Fig. 4l). Meanwhile, these lamellae show variation in thickness and wavy extinction indicating that these crystals were subjected to some strain.

The microgranite dykes are fine to mediumgrained and mainly composed of quartz, plagioclase, with subordinate amounts of orthoclase, microcline and biotite (Fig. 4m). The zircon, apatite and iron oxides are the main accessories.

Geological mapping using remote sensing data

Remote sensing analysis was carried out to discriminate the different rock units covering the study area, to delineate the hydrothermally altered rocks along shear zones and to produce more

Fig. 5.

a) 5/7 band ratio image,

detailed (1 :20,000) image map. Band ratio and fusion techniques are the main image processing techniques carried throughout this study. Landsat-7 ETM+ image of path 1 74 and row 44 acquired in 1 611 -2001 and the scanned B&W aerial photograph scale 1 :40 000 were digitally analyzed using ENVI v.3.4 image processing software. ETM+ scene has eight broad spectral bands detect visible (band-1 , 2, 3), NIR (band-4), SWIR (band-5&7), TIR (band-6) and panchromatic (band-8) bands.

Several studies applied the band ratio technique for lithologic mapping and rock unit discrimination.

The band ratio is prepared simply by dividing the DN values of each pixel in one band by the DN values of another band [1 2]. During present work, the 5/7, 4/5 and 3/1 band ratio images were firstly generated to recognize and differentiate between the carbonates, Fe-silicates and iron minerals, respectively. Then, the information contained in these three band ratio images were integrated into one false color composite ratio image (5/7:R,4/5:G & 3/1 :B). The false colour composite ratio image was generated from a combination of the three band-ratio images in red, green and blue. On the 5/7 band ratio image (Fig. 5a) of Wadi Um Arka area, the altered rocks within shear zones show bright image signature, whereas hornblende schists and granitic rocks appear with dark image signature. Moreover, gabbros and intermediate rocks show gray image signatures. On other hand, the 4/5 band ratio image (Fig. 5b) shows that gabbros and granitic rocks have bright image signature, whereas intermediate rocks (metaandesites and andesitic metatuffs) have dark image signature. Nearly all rock units exposed in the study

b) 4/5 band ratio image,

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c) 3/1 band ratio image.

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

a) FCC-ratio image (5/7, 4/5, 3/1 ), b) FCC-ratio image (7/5, 5/1 , 5/4*3/4), c) Fused image of Wadi Um Arka area.

Fig. 6.

Fig. 7.

area have gray image signature on 3/1 band ratio image (Fig. 5c) favoring that different concentrations of iron minerals occur within these rock units.

The false colour composite ratio image (5/7:R,4/5:G & 3/1 :B) of Wadi Um Arka area (Fig. 6a) shows that the altered rocks have rose, orange and yellowish image signatures, whereas the olivine gabbros and anorthosites have yellowish and light green image signatures. Also, the intermediate rocks and uralitized pyroxene gabbros have dark red image signature. This FCC-ratio image failed to discriminate clearly between basic and acidic rocks, where hornblende schists, anorthositic gabbros and granites have similar green image signatures.

Accordingly, another FCC ratio image (5/7:R, 5/1 :G & 5/4*3/4:B) was integrated to discriminate between these units. On this FCC ratio image (Fig. 6b) the basic and acidic rock units have dark blue and dark green image signatures respectively.

The fusion technique between multispectral and panchromatic data has been successfully used in several applications. The false coluor composite ratio image (5/7, 4/5 & 3/1 ) of Wadi Um Arka area

Geological map of Wadi Um Arka area, South Eastern Desert, Egypt.

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt was merged with the geometrically corrected panchromatic high spatial resolution scanned aerial photograph by using Hue, Saturation, Value (HSV) merger to produce more detailed (1 :20,000) image map. The resultant fused image (Fig. 6c) was used to delineate the hydrothermal alteration zones as well as the different rock units covering Wadi Um Arka area. Using the obtained fused image, more detailed geologic map of scale 1 : 20,000 was traced (Fig. 7).

Geochemistry

A total of 1 9 representative samples (1 0 from the granitoid rocks and 9 from the gabbroic rocks) from Wadi Um Arka area were chemically analyzed for major oxides and some trace elements. The chemical analyses were carried out in Laboratories of Nuclear Materials Authority, Cairo. The chemical data and calculated CIPW norms are listed in Tables 1 & 2.

Geochemistry of the granitoids

Harkerâ&#x20AC;&#x2122;s variation diagrams (Fig. 8) reveal general increase of K2O with increasing SiO 2 in both older and younger granites. The remainder major oxides (TiO 2, Al 2O 3, MgO, FeO and CaO) show negative relation with SiO 2, which can be attributed to fractional crystallization processes. Moreover, fractional crystallization is supported by the gradual decrease of normative anorthite (An) content from older granites to muscovite granites. However, the older granitoids are Na-rich (average Na 2O/K2O value = 1 .90) whereas the younger granites are Krich (average Na 2O/K2O value = 0.95 for biotite granites and 0.89 for muscovite granites). Plotting of the mafic index values versus the felsic index values [1 3] for the studied granitoids (Fig. 9a) shows that the plots of older granites indicate intermediate to acid differentiation, while the younger granitoids have strongly acid differentiation.

Petrochemical classification and nomenclature The granitoid rocks under investigation can be classified by using CaO-Na 2O-K2O ternary diagram [1 4] and Q versus P multicationic diagram [1 5]. The older granitoid samples plot within granodiorite and tonalite fields, whereas those of younger granites occur in adamellite field (Figs. 9b&c). Moreover, on SiO 2 versus (Na 2O + K2O) diagram of Middlemost [1 6] and normative An-Ab-Or ternary diagram of

Barker [1 7] plots of older granitoids fall within tonalite and granodiorite fields, meanwhile all the younger granite samples plot in the granite field (Figs. 9d & 1 0a). Chappell and White [1 8] used the Mol. Al 2O 3/(CaO+Na 2O+K2O) ratio against Rb/Sr ratio to differentiate between I-type and S-type granites. On this binary diagram (Fig. 1 0b) all the studied granitoid samples fall within the I-type field, except two older granitoid samples and one biotite granite sample which lie at the boundary separating between the two fields of I-type and S-type granites.

Moreover, White and Chappell [1 9] as well as Liew et al. [20] used the Na 2O-K2O binary diagram (Fig. 1 0c) to discriminate between I-type, S-type and T-type granites. Plots of the all granitoid samples lie in the I-type granite field indicating igneous source materials for the studied granites, except one biotite granite sample and another of muscovite granites fall within the transition (T-type) field.

Magma type, tectonic setting and petrogenesis All plots of the investigated granitoids fall within the calc-alkaline field (Fig. 1 0d) on the AFM ternary diagram of Irvine and Baragar [21 ]. Moreover, Petro et al. [22] used the AFM ternary diagram to discriminate between compressional and extensional suites. Plots of the studied granitoids mostly have the trend of compressional suite. On Wright [23] alkalinity ratio (A.R) versus SiO 2 binary diagram (Fig. 11 a) all plots of the older granitoids fall within the calc-alkaline field, while those of the younger granites lie in the alkaline field reflecting the increase of alkalinity from older towards the younger granites. On the Al 2O 3/(CaO + Na 2O + K2O) versus Al 2O 3/(Na 2O + K2O) binary diagram of Maniar and Piccoli [24], all plots of the older granitoids of peraluminous nature, whereas the younger granites are of peraluminous to metaluminous nature (Fig. 11 b). The calculated CIPW norms (Table 1 ) support the obtained results. The older granitoid samples have normative corundum (C) indicating alumina saturation and peraluminous nature.

Meanwhile, the younger granitoid samples are divided between C-normative indicating peraluminous nature and no normative corundum but have (Di+Hy) normative suggesting silica saturation and metaluminous nature.

Batchelor and Bowden [25] have used the R1 R2 multicationic relation of De La Roche et al. [26] to discriminate between the different tectonic setting

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

Table 1. Chemical

analyses and CIPW norms for the granitiod rocks, Wadi Um Arka area.

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

Table 2. Chemical

analyses and CIPW norms for the gabbroic rocks, Wadi Um Arka area.

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

Fig. 8. Variation

diagrams of silica against some major oxides for the granitoid rocks, Wadi Um Arka area.

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of the analyzed granitoid rocks on A) The mafic index versus felsic index diagram of Nockolds [1 3], B) The CaO-Na 2O-K2O ternary diagram (after Archibald et al. [1 4]), C) The Q-P multicationic classification diagram of Debon and Le Fort [1 5], D) The SiO 2 versus total alkalis discrimination diagram of Middlemost [1 6].

Fig. 9. Plots

Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

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Plots of the analyzed granitoid rocks on A) The normative An-Ab-Or ternary diagram (after Barker [1 7]), B) Mol. Al 2O 3/(CaO+Na 2O+K2O) versus Rb/Sr diagram (after Chappell and White [1 8]) discriminating between I-type and S-type granites, C) The Na 2O versus K2O binary diagram (after White and Chappell [1 9], the T-type field (after Liew et al. [20]), D) The AFM ternary diagram (Irvine and Baragar [21 ], the extensional (1 ) and compressional (2) trends after Petro et al. [22]).

Fig. 10.

Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

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Plotting of the studied granitoids on A) The alkalinity ratio (A.R) versus SiO2 binary diagram of Wright [23], B) The Al 2O 3/(CaO+Na 2O+K2O) versus Al 2O 3/(Na 2O + K2O) diagram (after Maniar and Piccoli [24]), C) The R1 -R2 binary diagram (Batchelor and Bowden [25]), D) The modal Q-A-P ternary diagram (after Maniar and Piccoli [24]) discriminating between the different tectonic environments of granitoids.

Fig. 11.

Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt of granitoid rocks. Plotting of the studied granitoid samples on this diagram (Fig. 11 c) illustrates that the older granitoids were mainly emplaced during the pre-plate collision stage, while the younger granites were formed through both late-orogenic and syn-collision stages. On the modal Q-A-P ternary diagram of Maniar and Piccoli [24] both the older granite and biotite granite samples are continental arc granitoids, except one biotite granite sample that represent an overlap between the CAG and CCG (continental collision granitoids), (Fig. 11 d). Also, the muscovite granite plots are mostly located in this overlap area.

On the normative An-Ab-Or ternary diagram of Barker [1 7] all plots of older granites fall in a high water-vapour pressure region, more than 5 k-bars, while the plotted younger granite samples fall in a region of low to moderate water-vapour pressure ranging from 1 to 5 k-bars (Fig. 1 0a). Meanwhile, the normative Q-Ab-Or diagram of Tuttle and Bowen [27] illustrates that the older granites were emplaced under high temperature conditions, ranging from about 800 to 840ยบC, whereas the younger granites were emplaced under moderate temperature conditions ranging from about 760 to 800ยบC (Fig. 1 2a).

The older granites show relatively low K/Rb (1 81 .2-301 .5), high Ba/Rb (4.2-8.9) and low Rb/Sr (0.2-0.4) compared with the younger biotite and muscovite granites (K/Rb 31 6.9-528.5 and 225.9474.9, Ba/Rb 5.1 -6.8 and 3.8-7.3, Rb/Sr 0.4-0.9 and 2.6-6.1 respectively). The K-Rb binary diagram (Fig. 1 2b) shows that the all granitoid plots are located in the region between the mantle line (K/Rb = 1 000) given by Shaw [28] and the crustal line (K/Rb = 250) suggested by Taylor [29]. This suggests the derivation of the studied granitoids from the lower crust or upper mantle source.

The Rb-Sr binary diagram (after Condie [30]) shows that the older granites have emplaced at greater depth >30 km in the crust, whereas the younger granitoids have emplaced at relatively shallow to moderate depths between 20 and 30 km (Fig. 1 2c). Moreover, It is evident that the Rb/Sr ratio increases with differentiation from the older granites towards the younger biotite and muscovite granites, indicating more differentiated and more evolved granitic liquids that formed the later types.

Geochemistry of the gabbros

The three members of younger gabbros repre-

sent different phases of differentiation, formed by successive crystal fractionation. Generally, the average contents of SiO 2, Na 2O, K2O and P 2O 5 increase gradually from olivine gabbros to pyroxene gabbros, then rise sharply in the highly differentiated anorthosites. On other hand, the average content of MgO decreases from the more basic olivine gabbros through pyroxene gabbros, then decreases sharply in the anorthosites. The average contents of Cr, Co, Ni, Ba and V decrease markedly from olivine gabbros to pyroxene gabbros and then decrease sharply in the highly differentiated anorthosites.

The average values of differentiation index (D.I) increase markedly from the more basic olivine gabbros (1 4.31 ) to pyroxene gabbros (23.25) and anorthosites (36.54). On other hand, the values of solidification index (S.I) decrease markedly from the olivine gabbros (54.75) to pyroxene gabbros (37.51 ) and anorthosites (6.1 6). The wide ranges of both D.I and S.I values (36.54-1 4.31 & 54.75-6.1 6 res.) suggest that the parent magma, from which these gabbroic rocks were derived, subjected to high differentiation along successive fractional crystallization processes.

Petrochemical classification and nomenclature The TAS classification diagram [31 ] and R1 -R2 diagram [26] were used to classify the studied gabbros. On both diagrams, all plots of gabbros fall within the olivine gabbro and gabbro fields (Figs. 1 3a&b). The plots of pyroxene gabbros and anorthosites are located in gabbro and gabbronorite fields, whereas those of olivine gabbro fall within olivine gabbro filed. All plots of olivine gabbros, pyroxene gabbros and anothosites on the An-Or-Ab normative diagram [32] are located in the mafic gabbro field (Fig. 1 3c).

Magma type, tectonic setting and petrogenesis Plotting of the studied gabbros on the total alkalis versus silica diagram of Irvine and Baragar [21 ] favors that the three members of younger gabbros were derived from subalkaline magma (Fig. 1 3d). Meanwhile, on the AFM diagram of Irvine and Baragar [21 ] differentiated the studied rocks as the tholeiitic (Fig. 1 4a). Yet the anorthosite samples fall in the calc-alkaline field, near and at the boundary separating between the two fields. Also, on the SiO 2-FeOt/MgO binary diagram of Miyashiro [33] all the plotted samples of the different gabbroic

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

Plots of the granitoid samples on A) The normative Q-Ab-Or ternary diagram of Tuttle and Bowen [27], B) The K-Rb variation diagram (Shaw [28]). The average crustal K/Rb ratio after Taylor [29], C) The Rb-Sr binary diagram. The dashed lines referring to the crustal thickness (after Condie [30]).

Fig. 12.

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of the studied gabbroic rocks on A) The TAS classification diagram (after Wilson [31 ], and B) The R1 -R2 diagram of De La Roche et al. [26], C) Plots of gabbros on the normative An-Or-Ab diagram of Hietanen [32], and D) The total alkalis versus silica diagram (after Irvine and Baragar [21 ]).

Fig. 13. Plotting

Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

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Plots of gabbros on A) The AFM ternary diagram (after Irvine and Baragar [21 ]), B) The SiO 2-FeOt/MgO binary diagram (Miyashiro [33]), C) The MgO-FeOt-Al 2O 3 diagram (after Pearce et al. [36]), D) The Cr-Y discrimination diagram (after Pearce [37]).

Fig. 14.

Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt

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Abdel Rahman et al. Plutonites of Wadi Um Arka, Egypt members lie in the tholeiite field (Fig. 1 4b).

Two models were proposed for the geotectonic environment and magma sources of the younger gabbros, the tensional environment or rift-related [1 , 34] and the subduction-related or island-arc environment [35]. On the AFM diagram, Petro et al.

[22] defined two tectonic trends discriminating between the compressional and extensional suites (see Fig. 1 4a). Plots of all gabbro members are found parallel to the compressional trend indicating their origin in compressional environment (subduction-related or island-arc environment). The MgO-FeOt-Al 2O 3 discrimination diagram [36] reveals that these gabbros were formed within island-arc and active continental margin environment (Fig. 1 4c). Plotting of the studied gabbros on the Cr versus Y discrimination diagram [37] favors the arc-related tectonic setting for these younger gabbros. Plots of all gabbro members fall within the field of volcanic-arc basalts (Fig. 1 4d).

CONCLUSIONS Detailed geological mapping of the area of Wadi Um Arka was carried out with remote sensing analysis discriminated the different rock units covering the area, delineated the alteration zones throughout shear zones and produced more detailed (1 :20,000) fused image map. The outcrops include the island-arc assemblage, syn-tectonic granites, late to post-tectonic granites and younger gabbros. Two main shear zones cut across the area, NW-trending shear zone and ENE-trending shear zone. Three effective major tectonic trends NW-SE, ENE-WSW and NE-SW are recorded. The syn-tectonic granites are mainly granodiorite with minor tonalite. They are calc-alkaline, peraluminous nature and sodic affinity. They have emplaced during pre-plate collision stage in continental arc regime under high water-vapour pressure (>5 k-bars), compressional stresses and high temperature (800-840ยบC) at greater depths more than 30 km.

The late to post-tectonic granites are typically monzogranites (biotite granites and muscovite leucogranites). The biotite granites have calcalkaline to alkaline, metaluminous to slightly peraluminous nature and potassic affinity. They have emplaced during late-orogenic regime in compressional suite. The muscovite leucogranites possess alkaline, peraluminous to slightly metalu-

minous and potassic affinity. These granites have emplaced during syn-collision stage. Both biotite and muscovite granites were formed under relatively low to moderate water-vapour pressure (1 -5 k-bars) and temperature (760-800ยบC) conditions at moderate depths (20-30 km). The muscovite granites have emplaced comparatively under the lowest conditions of water-vapour pressure and temperature as well as at the shallowest depth.

The younger gabbros comprise olivine gabbros, pyroxene gabbros and anorthosites. These gabbroic rocks represent different degrees of differentiation, formed by successive fractional crystallization processes. During earlier stages, the olivine gabbros and subordinate pyroxene gabbros were formed by crystal settling of Mg, Fe and Tibearing minerals. As crystallization progressed, the residual magma became very poor in Mg, Fe and Ti and more enriched in Al, Si, Ca and Na. The highly differentiated anorthosites were derived from the residual magma during the later stages of crystallization. These gabbros have been derived from subalkaline basic magma exhibiting tholeiitic nature with mild calc-alkaline tendency. They were emplaced in compressional subduction-related environment having characters of the island-arc and active continental margin.

ACKNOWLEDGEMENTS The authors are grateful to Prof. Dr. M. M. Hassaan, Faculty of Science, Al-Azhar Univ., for his kind assistance and constructive criticism to prepare the present study.

TRANSPARENCY DECLARATION The authors declare no conflicts of interest.

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ISSN: 2084-3577

TMKARPIŃSKI PUBLISHER

EARTH SCIENCES

Journal of Biology and Earth Sciences ORIGINAL ARTICLE

Petrological characteristics of the metamorphosed metaintrusives in amphibolite and granulite facies of the Kandalaksha part of Lapland Granulite Belt (Kola Peninsula, NW Russia) Miłosz A. Huber Optical and Electron Microscopy Laboratory, Geology and Lithosphere Protection Department, Maria Curie Sklodowska University, Lublin, Poland

ABSTRACT

This paper discusses the location and the relationship of metaintrusives and metasediments, which are located on the Kandalaksha region in Lapland Granulite Belt (LGB) of Kola Peninsula at the White Sea. Collected materials was made petrographic analysis, used macroscopic and microscopic optical techniques (including CL) and electron (with adapter EDS), also made a numerous analyzes by spectroscopic (IR, Mossbauer) and X-ray technique of rocks and separated minerals. As a result of these studies the LGB is constructed by two complexes rocks: amphibolite and granulite rocks as well as mataintrusive (gabbroide) and extrusive-lavas rocks representing andesite basalt volcanism with sediments and tufs. These rock complex have secondary minerals association which are more young diaphoretic processes near prehnite-albite-calcite mineral veins. These rocks are tectonic sliced and overlap at each other. The structure of LGB is composed mainly of amphibolite and granulite 2.4-2.8 Ga, which were created in the collision zone between the Kola and Byelomorian blocks. The complex originally sedimentary-volcanic rocks with gabbroide intrusions was heavily tectonically rebuilt and metamorphosed. These rocks are exposed at present in two places: on the border of Russia, Finland and Norway-in Lapland and in the Kandalaksha area, where they are strongly reduced. This paper is devoted to geology of the Kandalaksha part of the Lapland Granulite Belt with an interpretation of the structure of individual cells and petrographic analysis of rock types from which it is built.

Key words: Metaintrusives; Metasediments; Ortogranulites; Gneisses; Lapland Granulite Belt; Kola Peninsula; Kandalaksha.

J Biol Earth Sci 201 3; 3(2): E39-E46 Corresponding author:

Miłosz A. Huber Optical and Electron Microscopy Laboratory, Geology and Lithosphere Protection Department, Maria Curie Sklodowska University, 20 -71 8 Lublin, 2cd Kraśnicka rd., Poland, E-mail: mhuber@umcs.lublin.pl Original Submission: 02 October 201 3; Revised Submission: 07 November 201 3; Accepted: 1 5 November 201 3 Copyright © 201 3 Author(s). Journal of Biology and Earth Sciences © 201 3 Tomasz M. Karpiński. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://www.journals.tmkarpinski.com/index.php/jbes or http://jbes.strefa.pl e-mail: jbes@interia.eu Journal of Biology and Earth Sciences, 201 3, Vol 3, Issue 2, E39-E46

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia)

INTRODUCTION On the Kola Peninsula near Kandalaksha the rocks of Lapland Granulite Belt are present in the Kandalaksha’n Tundra Mts. These are metamorphosed to amphibolite and granulite facies sedimentary and intrusive rocks. Among the garnet amphibolite are present also feldspar-garnet gneiss and ilmenite-rutile-titanite-plagiocklase-garnet gneisses (metagabbroides) [1 -5]. This are old collision zone between Kola and Belomoryan block, strongly tectonic rebuilt and metamorphosed by in amphibolite and granulite facies [6-8]. Feldspar-garnet gneiss outcrop near the northern Zhielieznaya Mt. and eastern part of the study area at the northern slopes and Sriednyj Myss & Siennaya Kurtiazhnaya Mts. Ilmenite-rutile-titanite-plagiocklase-garList of the studied feldspar – amphibole gneisses with method of analysis.

Table 1.

net gneisses occur among metamyllonites around the northern peak of Zhielieznaya Mt. are metamorphosed in amphibolite facies, and around the peak of Siennaya Kurtiazhnaya and Sriednyj Myss Mt. are metamorphosed in granulite facies.

MATERIALS AND METHODS These rocks were studied using a polarizing microscope in transmitted and reflected light, electron microscopy with EDS, XRD pattern analysis and infrared absorption spectroscopy (Fig. 1 ).

These analysis was made at the Faculty of Geology, Geophysics and Environment Protection in University of Science and Technology (AGH) in Krakow, Poland (EDS analysis was make in Geology Institute in Jagiellonian University in Krakow and in the Optical and Electron Microscopic Laboratory, In Earth Science and Spatial Protection Faculty at Maria Curie-Skłodowska University in Lublin, Poland)

RESULTS AND DISCUSSION Feldspar-amphibole gneisses

Identification of samples of these rocks, with the methodology used their research is presented in Table 1 . These gneisses are yellow and light gray,

Fig. 1.

Scheme of localization of studied rocks in Kandalakshan part of LGB.

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia)

Macro (a) and microphotographs in polarized light of feldspar â&#x20AC;&#x201C; amphibole gneisses: feldspar, plagiocklases, amphiboles and chlorites (crossed pollars, b), two type of plagiocklases and chlorites (Nx, c), rutile with titanite, apidote and garnets (Nz, d). Microphotograph in reflected light of garnet with rutile, ilmenite and titanite inclusions (e, f).

Fig. 2.

with visible garnets, amphibole laminae (Fig. 2a), which have chloritization places. Larger concentrations of chlorites give it a greenish color. These gneisses have grano- lepidoblastic structure, directional gneiss or pencil texture. They consist of amphibole (1 -59%), pyroxene (0-7%), plagioclase (0-51 %), garnets (3-28%) and accessory minerals.

In thin section background plagioclase crystals build

rock arranged in a random, between which there are aggregates of mafic minerals forming the laminae.

Idioblasic plagioclase are often polysynthetic twinned (Fig. 2b, c), about the size of up to 1 -2 mm, sometimes subject to strong sericitization, which makes it impossible to identify them. In intersticia of these minerals are amphibole, chlorite and epidote

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia) group minerals. The size of a match or even exceed the garnet blasts. Surrounded by finer individuals, distributed in a random manner, forming a rock background. Usually, there are sericitizate varying degrees. They are about 60% vol. of rocks. The ratio of these minerals to leucocratic components is highly variable. Analysis of the nature of the plagioclase is optically identified: basic (labrador An 78-80%) and acid (oligoclase). Quartz is present to in the studied gneisses (average about 1 3.7% vol.) Is randomly distributed between the blasts feldspar (Fig. 2b). Amphibole are represented by common hornblende (up to 56% by volume) mixed with tschermakite which have a pleochroism by: greenish (β) and colorless (α). There are mostly idiomorfic, reaching the size of a few mm. Often fuse together along the (011 ) or (11 0). Solid inclusions rich in ilmenite and iron oxides. Hornblende accompanied by chlorite and epidote. These products are distributed in the transformation and are filling microcracks along the cleavage planes.

Epidote (rich in klinozoizite partice) is in the form of single crystals within the feldspar - quartz aggregates. Creates sometimes with chlorites (Fig. 1 d) a large accumulations. In some samples near amphibole are present pyroxene, of several percent by volume of rocks. Clinopyroxenes are mainly represented by diopside and orthopyroxsene - hipersten.

They are, amfibolization. Hyperstene is surrounded by garnets. Biotite occurs in the common hornblende. The share of this mineral in the rock is negligible (less than 1 % vol.). Garnets (rich in almandine molecule containing grossulare) in these rocks developed of idioblastic form, with a diameter of a few millimeters [9]. They contain inclusions of quartz, titanite, rutile. The microphotographs (Fig. 2d, e, f) shows that these minerals were repeatedly regenerated. Evidence of this arrangement in them, as well as the shape of the inclusions. The amount of these minerals in the rock at an average of 20% vol. Participation molecules rich in calcium points mainly in samples of metamyllonites. These rocks are present in the oxides of titanium (Fig. 2d, e, f) and iron and copper sulfides. They are: rutile (rich in vanadium impurity), titanite, ilmenite, pyrite and chalcopyrite. Occasionally there is also subside.

Ilmenite-rutile-titanite-plagiocklase-garnet gneisses (metagabbroides)

They are bright yellowish, yellow-grey, with visible garnets and amphiboles, plagioclases or pyro-

xenes (in the vicinity of granulites, Fig. 3a). They are characterized by the of grano-lepido-nematoblastic structure, incoherent or gneissic texture, highlighted by the distribution of garnets and amphibole arranged in the form of laminae. They are present: rutile, titanite, ilmenite (3-1 4%), plagioclase (6-61 %), garnets (6-37%), pyroxene (0-25%), amphibole (0-1 4%). In thin section, these rocks are composed of plagioclase twinning polysynthetic arranged in a random manner, forming a background rocks (Fig. 3b). There are arranged in a random manner in the rock, between which there are concentration of femic minerals. The chemical composition determined basis on the EDS shows two types of plagioclase. The first is a basic plagioclase, strongly sericitizing, the second - acid, is most common in rocks affected by the saussuritizating (in close proximity to the prehnite - feldspar - calcite veins). They are accompanied by titanium minerals: rutile, titanite, ilmenite (Fig. 3d, e, f), and magnetite. The rocks rich in pyroxene titanium minerals are represented by ilmenite, brookite and rutile. Ilmenite forms a rim around grains garnets. In the immediate vicinity of veins are visible in these rocks: hematite, maghemite, pyrite and chalcopyrite. They are present near plagioclases aggregates, sometimes constitute about 1 0% by volume of rocks.

Observation in reflected light shows that ilmenite from metagabbroides have the zonal construction and is rich in inclusions of ülvite. It is a structure decays of ilmenite. In some crystals of titanite in octahedral position observed increased amount of aluminum, which may be associated with increased pressure during the crystallization of the mineral aseemblage (ilmenite disintegration and amphibolization of pyroxene). Pyroxene are represented mainly by diopside (IR bands: 536, 562, 870, 988, 2425, 1 067 cm -1 ) and hipersten, representing approximately 25% by volume of rocks (opx 1 8.52 and cpx 6.35 % vol). They are present in theserocks, which are in the vicinity of granulite. Near pyroxene are amphibole whose participation ranges from 0-1 5% by volume. They are common hornblende (IR bands: 456, 501 , 658, 693, 982 cm -1 ) or high titanium hornblende (IR bands: 956, 982 cm -1 ), ksenomorphic, oval, surrounded by titanite. Titan in the mineral pyroxene comes from the transformation. Near amphiboles is epidote (IR bands: 456cm -1 and 956cm -1 ). Epidote and chlorite (Fig. 3c), are associated with the activity of hydrothermal processes (neighborhood prehnite – albite – calcite

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia)

Macro (a) and microphotographs in polarized light of high titanium rocks: titanite near garnets n sample (Nx, b), ilmenite, titanite and rutile (Nx, c), atolic garnet, plagioclases and biotites (Nx, d). Microphotographs in reflected light of rutile with titanite near garnets cistalls (Nx, e), rutile with titanite with garnet and hematizing pyrite in upper part of photograph (Nx, f).

Fig. 3.

veins) [1 0]. In the sample 22eKK03 were found klinozoisyte and epidote, which are arranged in a random manner between plagioclase plaques.

Association of prehnitowo epidote - albite - calcite vein confirms the presence in the bar (found in mikroinklousion in the mineral barite). With these minerals coexist mica (Fig. 3d). It is a unit - biotite flogopite crystals disease associated with epidote and feldspars [11 ]. Combine these minerals are focused like pyroxene pseudomorphoses. If that is

the result of the replacement of pyroxene by mica, which have been referred to as chlorite. Garnets are oval blasts, surrounded by titanium minerals (7-30% by volume). They have glomeroblastic form. In aggregates of garnets are also present atolle form (Fig. 3d). Some of them make pseudomorphoses the titanium augite. The titanium and iron it became part of ilmenite. In the less metamorphosed metagabbr is high titanium hornblende with titanite. It is a solid solution involving molecules almandine,

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia)

Macro (a) and microphotographs in polarized light of orhogranulites: garnets with ilmenites (Nx, b).

Microphotographs in reflected light of garnets with ilmenites (Nx, c), ilmenite, rutile titanite and broockite (d), twinning titanomagnetite with ilmenites and chalcopyrite (1 N, e), pyrite, ilmenite, titanite and garnets (1 N, f).

Fig. 4.

knorringite and spessartine (XRD) [11 ]. Quartz in these rocks present in an amount of 9-30%. High levels of this mineral in the sample 1 6KK03 might be the result of silicified rocks. This mineral forms single crystals in rocks of secondary processes (saussurityzation in the vicinity of the plagioclase prehnite - calcite veins). Moreover, these rocks are: zircon with a small admixture of hafnium (SEM-EDS, sample 1 7KK02), oxygen apatite (SEM -EDS, sample 22eKK02), and more late associa-

tion: prehnite and calcite veins [1 0].

White orthogranulites

These rocks are yellowish-pinkish-greenish, with large of up to a few mm pyroxene and garnets blasts, inherent in the background plagioclase quartz (Table 2, Fig. 4a). The structure of these rocks is grano-lepido-nematoblastic, chaotic texture or pattern (Fig. 4a). They consist of: pyroxene (to 1 5%), garnets (20-50%), plagioclase (20-40%),

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia) Table 2.

List of the orthogranulites with method of analysis.

quartz (0-5%), amphiboles (0-1 5%) and ore minerals, especially rich in titanium (to 8%). The rocks of the most dominant melanocratic minerals. In assessing the color index does not take into account the garnets, which are in some samples of these rocks as much as 50% by volume. In thin section, the white orthogranulites is leucocratic rock background with glomeroblastic clusters melanocratic minerals (Fig. 4b), which are represented by randomly distributed crystals of plagioclase (labrador). In some specimens of advanced secondary processes, there was also the presence of albite and oligoclase (these processes is probably a result of propagation of carbonate fluids migration) [1 0].

Feldspars sometimes occur between a few crystals of quartz. Near these minerals are a xenomorphic garnets. The diameters of garnets blasts in some cases exceed a few mm. There are distributed randomly in the background pyroxene and feldspar.

There is a almandine-pyrope solid solution with addition of grossular (grossular unit is a result of secondary processes) [11 ]. These minerals are developed in the form of granoblastic and atollic (Fig. 4b) surrounding plagioclase and quartz. They constitute 1 5-50% by volume of rocks. Have inclusions of rutile, ilmenite, quartz and hercinite (Fig. 4c, d). In some samples rich in titanium, garnets have accompanied by ilmenite and rutile. Are also present a garnets, which are pseudomorphoses after the pyroxene. These garnets in granulites are solid solution of almandine and spessartine mixed with particles pyrope and grossulare. The presence of the molecule associated with abundance of calcium. The largest share of these minerals is al-

mandine and pyrope molecule containing crystallized later grossulare present in those individuals who have been subject of regeneration. In these minerals are also spinels inclusions (hercinite).

Large crystals of pyroxene, occurring in the rock is ortho-and clinopyroxenes, represented by diopside and hipersten, dominated orthopyroxenes. In some of these rock samples collected in the vicinity of veins, pyroxene are amfibolization. There are also remnants of augite, surrounded by a crown of reaction. In all of the analyzed samples in pyroxenes states high content of calcium. This is due to the presence of these large amounts of plagioclase rocks and garnets. In the sample 1 8KK03 (1 b) are small amount of tellurium (0.78 wt%), chromium (0.75% wt in sample 1 4KK03, 1 -1 ) and potassium (0.48% wt.). Occasionally in these rocks near pyroxene, usually in the vicinity of mineral veins is common hornblende (XRD, SEM-EDS) present. In comparison with the total number of components mafic (pyroxene, amphibole, garnet) in white granulites, part pyroxene is significant with the exception of rocks with advanced secondary processes.

It is found that in these rocks, there are two types of amphibole: rich in titanium hornblende and amphibole calcium - magnesium (tremolite), characteristic of epidote â&#x20AC;&#x201C; amphibole facies. Are accompanied by secondary minerals: acid plagioclase, epidote, carbonate and chlorite. In some variations of the rocks in the vicinity of the vein is visible enrichment epidote and mica. Micas are up to 5% by volume. Occasionally rocks adjacent to amphibole. These minerals are found with an electron microscope with EDS attachment. Analyzed biotite

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Huber Petrological characteristics of metaintrusives of the Lapland Granulite Belt (Kola Peninsula, Russia) is rich in magnesium (11 .11 % wt .) and iron (1 7.72% wt.), with the addition of calcium (0.64% wt.) and potassium (0.23% wt.). Chlorite and epidote in orthogranulite are located in the areas of contact veins near the blasts of pyroxene and hornblende acting in two generations: calcium (group zoisite clinozoisite) and rich in calcium, iron with small amounts of titanium and magnesium (epidote). Ore minerals are present in these samples frequently, sometimes up to 8% by volume of rocks. In the zones of occurrence of secondary hornblende appears ilmenite, rutile and titanite (Fig. 4f). Ilmenite in some granulites has twinning along [0001 ] (Fig. 4e), often overgrow on garnets (Fig. 4d). It is found also chromite, brookite, pyrite and chalcopyrite. Pyrite oxidation conditions by the action goes into hematite (Fig. 4f).

CONCLUSIONS Feldspar - amphibole gneiss rocks have multistage origin. Presence of the garnet indicates that they can be metemophoused aluminum-rich sediments. Minerals of titanium, phosphorus, and the nature of some plagioclase indicates that the primary could be gabbroide rocks which have undergone changes several times (weathering), it have metamorphosed. In addition, the presence of close metamyllonite contributes to the enrichment of slate in other mineral association, connected with wires filling tectonic zone, or low-facies metamorphic minerals associated with dynamic effects.

The ilmenite-rutile-titanite-plagiocklase-garnet gneisses rocks are metagabbro which are present in both among type of rocks: granulites and amphibolites. Contain pyroxene-garnet rocks, which appear to be xenoliths from the Earth's mantle. These rocks are rich in minerals such as spinel, titanium, and iron, which are present in both the oxide and in the silicate minerals (pyroxene, garnet, amphibole). These rocks sometimes is myllonitizing. The mylonitization of these works contributed to their silification.

Granulite facies rocks are mainly magmatic origin (metagabbroides) rich in titanium, with a large share of supracrustal composition. This is evidenced by minerals such as spinels, chromites, titanium oxides and iron, and a large amount of calcium, which is headquartered in plagioclase, garnets and pyroxenes. These rocks bear the effects of diaphtoretic processes which locally indicate after the amfi-

bolization and biotitization of pyroxenes, garnets, and the appearance of chlorite – albite associations related to the impact of prehnite – albite – calcite veins.

TRANSPARENCY DECLARATION The author declares no conflicts of interest.

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